CN101487005A - Gene of human vascular endothelial growth factor monoclonal antibody and use thereof - Google Patents
Gene of human vascular endothelial growth factor monoclonal antibody and use thereof Download PDFInfo
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- CN101487005A CN101487005A CNA2008101204301A CN200810120430A CN101487005A CN 101487005 A CN101487005 A CN 101487005A CN A2008101204301 A CNA2008101204301 A CN A2008101204301A CN 200810120430 A CN200810120430 A CN 200810120430A CN 101487005 A CN101487005 A CN 101487005A
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Abstract
The invention relates to heavy chain and light chain variable region genes of a monoclonal antibody of a human vascular endothelial growth factor, a polypeptide that is encoded by the genes, a carrier that contains the genes, and application of the genes and the polypeptide in the preparation of a clinical testing agent of VEGF-related tumors. The invention utilizes gene engineering means to firstly obtain the heavy chain and light chain variable region genes of the monoclonal antibody from a hybridoma cell strain of the monoclonal antibody with VEGF165 secretion, the heavy chain variable region gene has the nucleotide sequence with SEQ ID NO of 1, the light chain variable region gene has the nucleotide sequence with SEQ ID NO of 3, and the genes are combined with scFv small molecule antibody and cloned into a pET28a expression carrier, have high-efficiency expression in colon bacillus and generate protein that has reserved antibody combination capability and VEGF165 action function. Through the modification with gene engineering techniques, the invention establishes the foundation for biological missile construction, has ultra-convenient antibody production and greatly lowered cost, and is favorable for industrial production.
Description
Technical field
The present invention relates to the heavy chain and the chain variable region gene of human vascular endothelial growth factor monoclonal antibody, by the polypeptide of described genes encoding, contain carrier and the described gene and the application of polypeptide in the clinical detection reagent of the relevant tumour of preparation VEGF of described gene.
Background technology
VEGF is a kind of powerful and can produce the cytokine of various biological effect, it is the class glycoprotein that separation and purification is come out in Niu Chuiti folliculus stellate cell nutrient solution such as Ferrarra in 1989, be a member of Thr6 PDGF BB family, molecular weight is 34-45kDa, the sequence high conservative is distributed widely in the tissues such as brain, kidney, liver, spleen, pancreas and bone of humans and animals.People VEGF is the homodimer that is formed by two glycoprotein chains, because gene transcription level cut mode difference, VEGF has 5 kinds of hypotypes, VEGF at least
121, VEGF
145, VEGF
165, VEGF
189 and VEGF
206, VEGF wherein
121And VEGF
165Function is the strongest and secrete in the extracellular with the soluble proteins form.
Monoclonal antibody is made up of two identical heavy chains and light chain.Each bar heavy chain (VH) and light chain (VL) all are divided into constant region and variable region two parts.Wherein, VH and VL form antigen-binding site jointly.The specificity of antibody is determined by this position just.VH and VL are about 110 amino acid, account for 1/2 and 1/4 of weight chain respectively.Real and antigenic determinant space conformation complementary are 3 amino acid variation zones (CDR) greatly in antigen-binding site.Relative with the variable region is the skeleton district, and its aminoacid sequence is conservative relatively.ScFv is spliced by VH and VL and a flexible amino acid short peptide.
Abroad have report from phage display library screen VEGF antibody (
Baca MDeng J BiolChem.1997Nov; 272 (16); _ 10678-84.), but because this antibody is not cross through immunity, general avidity is supported, and is not suitable as clinical detection reagent.Domestic have the report that the strain of VEGF antibody hybridoma cell sets up (
Shi QiDeng,
Cell and molecular immunology magazine, 2008 02 phases), but also do not have the report of genes involved sequence up to now.
Summary of the invention
In order to seek at the clinical detection reagent that is used for the relevant tumour of VEGF, strain is applied to sudden change easily in the scale operation at antibody hybridoma cell, the cell strain instability, lose easily, many shortcomings such as cost dearly, using gene engineering means of the present invention, a kind of human vascular endothelial growth factor monoclonal antibody chain variable region gene and heavy chain variable region gene and expression product thereof are provided, and the genetic engineering antibody fragment of having assembled the scFv type, make the extensive cheap antibody of producing become possibility.
Another object of the present invention is that human vascular endothelial growth factor monoclonal antibody and scFv antibody fragment thereof are used for VEGF in preparation
165Application in the detection reagent of relevant tumor disease.
In order to realize first above-mentioned purpose, the gene of human vascular endothelial growth factor monoclonal antibody of the present invention, this gene comprises heavy chain variable region gene and chain variable region gene, heavy chain variable region gene has the nucleotide sequence of SEQ ID NO:1, and chain variable region gene has the nucleotide sequence of SEQ ID NO:3.
By the polypeptide of above-mentioned genes encoding, this polypeptide has the aminoacid sequence of SEQ ID NO:2 and the aminoacid sequence of SEQID NO:4.
A kind of expression vector, this expression vector contain the chain variable region gene of the nucleotide sequence of the heavy chain variable region gene of nucleotide sequence of SEQ ID NO:1 and SEQ ID NO:3.
As preferably, this expression vector is pET28a-scFv.
The polypeptide that above-mentioned expression vector is expressed.
The production method of the polypeptide that above-mentioned expression vector is expressed, comprise the steps: to isolate the heavy chain variable region gene VH and the chain variable region gene VL of antibody from the strain of VEGF165 monoclonal cell, and assembling scFv, scFv with assembling makes up prokaryotic expression carrier, transformed into escherichia coli, make up genetic engineering bacterium, and abduction delivering, to expression product separate, purifying and renaturation.
As preferably, the production method of the polypeptide that above-mentioned expression vector is expressed comprises the steps:
1. from VEGF
165The variable region gene of monoclonal cell strain separation antibody:
Collect well-grown, can secretion of VEGF
165The hybridoma cell strain of monoclonal antibody extracts total RNA,
The design degenerate primer, heavy chain variable region gene VH employing primer VH Back (5 '-AGGTSMARCTGCAGSAGTCWGG-3 ') and primer VH For (5 '-TGAGGAGACGGTGACCGTGGTCCCTTGGCCCCAG-3 '); Chain variable region gene VL employing primer VL back (5 '-GACATTGAGCTCACCCAGTCTCCA-3 ') and primer VL For (5 '-GTTAGATCTCGAGCTTGGTCCC-3 '),
According to single stage method RT-PCR test kit increase respectively heavy chain variable region gene VH and chain variable region gene VL, adopt the RT-PCR method respectively, angle and get antibody variable region heavy chain variable region gene VH and chain variable region gene VL, the clone enters carrier pMD18-T respectively;
2. heavy chain variable region gene VH, the clone of chain variable region gene VL amplified production:
Design of primers is as follows:
VH P
1(5 '-ACCATG
GAATTCCTGCAGGAGTCTGGTGGCTTG-3 '), contain the EcoRI restriction enzyme site,
VH?P
2(5’-AGAGCCACCTCCGCCTGAACCGCCTCCACCTGAGGAGACGGTG-3’)，
VL?P
1(5’-CAGGCGGAGGTGGCTCTGGCGGTAGTGACATTGAGCTCAC-3’)，
VL P
2(5 '-GTGGTG
AAGCTTTCACTCGAGCTTGGTACCACCTC-3 '), contain the HindIII restriction enzyme site,
Adopt Over-lap PCR, one section flexible linker (G of design between VH and VL
4S)
3, VH-linker-VL clones in efficient prokaryotic expression carrier pET28a;
3. efficiently express, purifying, renaturation scFv:
PET28a-scFv transforms and enters BL21 (DE3), and the IPTG abduction delivering is collected the inclusion body precipitation, urea dissolving, Ni
2+-IDA His-bind affinitive layer purification albumen, dilution method and dialysis method are to the target protein renaturation, the albumen of purifying is diluted to renaturation buffer I, renaturation 18～30h, the renaturation buffer II that then albumen packed into, dialysis renaturation 10～15h, ultrafiltration pipe Centricon 30 protein concentrates, 13%SDS-PAGE detects, protein-20 ℃ preservation.
As preferably, above-mentioned renaturation buffer I selects 50mM Tris-HCl, 0.15mM NaCl, 2M urea, 0.5Mm reduced glutathion, 0.1mM Sleep-promoting factor B, pH8.0 for use, and final concentration is 100 μ g/ml, renaturation 24h.
As preferably, above-mentioned renaturation buffer II selects 50mmol/L Tris-HCl, 0.15mol/L NaCl, pH8.0 for use.
The present invention utilizes genetic engineering means at first from secretion of VEGF
165The hybridoma cell strain of monoclonal antibody in obtained the weight chain variable region gene of monoclonal antibody, and assembled the scFv small molecular antibody, be cloned in the pET28a expression vector, and can in intestinal bacteria, efficiently express, the protein of its generation has kept the ability with antibodies, can with VEGF
165Effect, and with traditional sense on hybridoma antibody relatively have following advantage: the scFv molecule is little, by the transformation of genetic engineering technique, combines with some other effector molecules easily, lays the first stone for making up biological missile; Hybridoma cell strain is not easy to preserve, large-scale production cost height, and genetic engineering antibody can adopt prokaryotic expression system, can produce antibody very easily, cost reduces greatly, is beneficial to suitability for industrialized production.Therefore the production of genetic engineering antibody is with the obvious advantage than hybridoma cell strain generation antibody, thereby can replace the conventional hybridization tumor cell strain to produce antibody.In addition, VEGF
165Genetic engineering antibody can be used for making VEGF
165Affinity chromatography medium be used for VEGF
165Purifying.
Description of drawings
Fig. 1 is the RT-PCR product agarose gel electrophoresis analytical results of VEGF165 antibody variable region VH and VL, and wherein swimming lane 1 is a standard protein; Swimming lane 2 is the VH gene, and molecular weight is greatly about 340bp; Swimming lane 3 is the VL gene, and molecular weight is greatly about 330bp; Swimming lane 4 is the VH-linker-VL gene, and size is about 750bp.
Fig. 2 is the assembling of VEGF165 scFv gene and the synoptic diagram of expression vector establishment.
Fig. 3 is the abduction delivering of VEGF165 scFv, the analytical results of purifying and evaluation, wherein (A1): standard protein; (A2): do not induce whole bacterial protein; (A3): induce the back whole bacterial protein; (A4): ultrasonic supernatant; (A5): ultrasound precipitation; (A6): purified product; (B1): Western blotting detects proteic expression.
Fig. 4 is the synoptic diagram that combines that non-competitive ELISA is measured scFv and VEGF165.
Fig. 5 is that competitive ELISA is measured the synoptic diagram that combines that scFv suppresses monoclonal antibody and VEGF165.
Fig. 6 be Western blotting detect scFv and VEGF165 combine wherein (A1): standard protein; (A2): purifying VEGF165 albumen; (B): mAb and VEGF165 combine combining of (C): scFv and VEGF165; (D): combine (as the negative control) of irrelevant IgG and VEGF165.
Embodiment
The present invention is further described below in conjunction with drawings and Examples.
Embodiment 1: from the variable region gene of VEGF165 monoclonal cell strain separation antibody
Collect well-grown, the hybridoma cell strain ABL-110 of energy secretion of VEGF 165 monoclonal antibodies
7Individual (this research department makes up, according to " Current protocol in immunology " Production ofMonoclonal Antibodies) extracts total RNA with TRIZOL reagent.
RT-PCR angles and gets the VH gene:
The method that provides with reference to (the Access RT-PCRSystem and Access RT-PCRIntroductory System) of Promega company: the single stage method RT-PCR test kit VH gene that increases respectively:
VH gene employing primer VHBack (5 '-AGGTSMARCTGCAGSAGTCWGG-3 ') and primer VHFor (5 '-TGAGGAGACGGTGACCGTGGTCCCTTGGCCCCAG-3 '),
The RT-PCR reaction is total up to 50 μ l systems: total RNA of 1 μ g, and 5 * AMV/Tf1 reaction buffer of 10 μ l, concentration is primer VH Back and each 5 μ l of VH For of 10pmol, concentration is the dNTP1 μ l of 500uM, the MgSO of 3mM
42 μ l; 5u AMV reversed transcriptive enzyme 1 μ l and 5uTf1 archaeal dna polymerase 1 μ.Reactions steps: 48 ℃ of reverse transcription reactions 45 minutes; 94 ℃ of sex change 30 seconds; Annealed 1 minute for 55 ℃; 68 ℃ were extended totally 35 circulations 1 minute.
RT-PCR angles and gets the VL gene:
VL gene employing primer VL back (5 '-GACATTGAGCTCACCCAGTCTCCA-3 ') and VL For (5 '-GTTAGATCTCGAGCTTGGTCCC-3 ').According to the single stage method RT-PCR of Promega company test kit amplification VL.50 μ l reaction systems are as follows: total RNA of 1 μ g, and 5 * AMV/Tf1 reaction buffer of 10 μ l, concentration is primer VL back and each 5 μ l of VL For of 10pmol, concentration is the dNTP1 μ l of 500uM, the MgSO of 3mM
42 μ l; 5u AMV reversed transcriptive enzyme 1 μ l and 5u Tf1 archaeal dna polymerase 1 μ.Reactions steps: 48 ℃ of reverse transcription reactions 45 minutes; 94 ℃ of sex change 30 seconds; Annealed 1 minute for 60 ℃; 68 ℃ were extended totally 35 circulations 1 minute.After reaction finished, 1% agarose gel electrophoresis detected.Result such as Fig. 1.
The clone of VH and VL gene amplification product:
Design of primers is as follows:
VH P
1(5 '-ACCATG
GAATTCCTGCAGGAGTCTGGTGGCTTG-3 '), contain the EcoRI restriction enzyme site,
VH?P
2(5’-AGAGCCACCTCCGCCTGAACCGCCTCCACCTGAGGAGACGGTG-3’)，
VL?P
1(5’-CAGGCGGAGGTGGCTCTGGCGGTAGTGACATTGAGCTCAC-3’)，
VL P
2(5 '-GTGGTG
AAGCTTTCACTCGAGCTTGGTACCACCTC-3 '), contain the HindIII restriction enzyme site,
The PCR product uses glue to reclaim the test kit purifying.Get carrier pMD18-T 1 μ l, PCR product 4 μ l, T4 ligase enzyme 1 μ l, 5 μ lT4 ligase enzyme damping fluids (2 times), 16 degree connect 18 hours.Connect product 10 μ l transformed competence colibacillus bacillus coli DH 5 alphas.Bed board, second day picking mono-clonal adopts bacterium colony PCR to identify, and delivers the order-checking of Shanghai Bo Ya company.Sequencing result is shown in sequence table SEQ ID NO:1 and SEQ ID NO:3.
The assembling of embodiment 2:scFv gene
For obtaining scFv, between VH and VL, add a flexible linker (G
4S)
3, for prevent the sudden change that induces one in amplification procedure, we adopt high-fidelity enzyme Pyrobest (Dalian is precious biological). and whole assembling synoptic diagram is Fig. 2, result such as Fig. 1.
First round PCR reaction amplification VH:
50 μ l PCR reaction systems are as follows: 10 * reaction buffer of 5 μ l; Concentration is the primer VH P of 10 μ M
1With VH P
22.5 the dNTP of μ l:4 μ l; 35 μ l H
2O; High-fidelity Pyrobest enzyme 0.5 μ l.95 ℃ of sex change 5min; 95 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 30s, totally 26 circulations; 72 ℃, extend 5min.
Second takes turns PCR reaction amplification VL:
50 μ l PCR reaction systems are as follows: 10 * reaction buffer of 5 μ l; Concentration is the primer VL P of 10 μ M
1With VL P
22.5 the dNTP of μ l:4 μ l; 35 μ l H
2O; High-fidelity Pyrobest enzyme 0.5 μ l.95 ℃ of sex change 5min; 95 ℃ of sex change 30s, 52 ℃ of annealing 30s, 72 ℃ are extended 30s, totally 26 circulations; 72 ℃, extend the 5min. rubber tapping and reclaim the PCR product.
Third round PCR reaction amplification VH-linker-VL:
50 μ l PCR reaction systems are as follows: 10 * reaction buffer of 5 μ l; Concentration is the primer VH P of 10 μ M
1With VL P
22.5 the dNTP of μ l:4 μ l; 35 μ l H
2O; High-fidelity Pyrobest enzyme 0.5 μ l, each 1 μ l of last two-wheeled PCR product is as template.95 ℃ of sex change 5min; 95 ℃ of sex change 30s, 52 ℃ of annealing 30s, 72 ℃ are extended 30s, totally 26 circulations; 72 ℃, extend the 5min. reaction.1% agarose gel electrophoresis, product is reclaimed in rubber tapping.
The enzyme of embodiment 3:scFv gene is cut digestion and is connected
Double digestion pET28a:
EcoRI and HindIII are the Takara product.10 * M reaction buffer of 2 μ l; The carrier pET28a of 10 μ l; Each 1 μ l of EcoRI and HindIII; 7 μ l H
2O.37 ℃ were reacted 6 hours.：
Third round amplified production among the double digestion embodiment 2:
10 * M reaction buffer of 2 μ l; The third round PCR product of 10 μ l; Each 1 μ l of EcoRI and HindIII; 7 μ l H
2O.37 ℃ were reacted 6 hours.1% agarose gel electrophoresis, product is reclaimed in rubber tapping.
Ligation:
The T4 ligase enzyme is the Takara product.The ligation damping fluid H of 2 μ l
2O; Last wheel enzyme is cut each 1 μ l of product; 16 μ l H
2O.16 ℃ were reacted 12 hours.
To get 10 μ l products and transform transformed competence colibacillus DH5 α. bed board, second day picking mono-clonal adopts bacterium colony PCR to identify, and delivers the order-checking of Shanghai Bo Ya company.
The abduction delivering of embodiment 4:scFv single-chain antibody in intestinal bacteria.
Clone's transformed competence colibacillus BL21 (DE3) that the picking order-checking is correct. the single colony inoculation of picking is in 3ml LB nutrient solution (containing 50 μ g/ml kantlex), cultivated 10 hours for 37 ℃, collect bacterium, being inoculated into 50ml by 1:100 contains among the same concentrations microbiotic LB, 37 ℃ when being cultured to 0D600=0.6, add IPTG (1mmol/L) and induce, cultivated 5 hours for 37 ℃, 13%SDS-PAGE detects expression. and result such as Fig. 3 clearly show that the expressed proteins molecular weight is about 27Kd.Mainly be present in the inclusion body.
Embodiment 5: the separation and purification of expression product and renaturation
4 ℃ of centrifugal 10min of 5000g/min collect thalline, with 5ml50mmol/L Tris-HCl, 0.15mol/L the resuspended precipitation of NaCl, ultrasonic on ice then (ultrasonic 5s, gap 5s, 160 times repeatedly), to cell suspension thickness no longer. the centrifugal 10min of room temperature 12000g/min, to collect respectively and go up cleer and peaceful precipitation. precipitation adds 2.5ml Binding damping fluid (50mmol/L Tris-HCl, 0.15mol/L NaCl, 8M urea, the 5mM imidazoles, pH8.0), the room temperature dissolving is 2 hours. and the centrifugal 10min of room temperature 12000g/min, collect supernatant. get Ni
2+-IDA His-bind resin1ml adorns post, the operational manual purifying protein that provides according to Novagen company.
Collect the albumen of Elution buffer solution elution, measure protein concentration, with renaturation buffer I (50mmol/L Tris-HCl, 0.15mol/L NaCl, 2M urea, 0.5mmol/L reduced glutathion, 0.1mmol/L Sleep-promoting factor B pH8.0) diluted protein to final concentration is 100 μ g/Ml. renaturation 24h, then albumen is adorned people's dialysis tubing, with renaturation buffer II (50mmol/L Tris-HCl, 0.15mol/LNaCl, pH8.0) dialysis renaturation 12h. ultrafiltration pipe Centricon 30 (Millipore, USA) protein concentrate .13%SDS-PAGE detects. protein-20 ℃ preservation.
Embodiment 6:scFv single-chain antibody combines determination of activity with VEGF165
1) non-competing ELISA method is measured:
10 μ g/ml VEGF165 (NaHCO
3PH9.6) 4 ℃ of wrapper sheets spend the night. and second day with (37 ℃ of 1% skimmed milk sealings, 2h). the scFv of serial dilution is hatched (37 ℃ respectively with VEGF165,2h). with anti-His monoclonal antibody (Novagen, USA) be one anti-, HRP link coupled goat anti-mouse igg is two anti-, and conventional ELISA detects (with reference to Current protocol in immunology unit2.1-2.3). result such as Figure 42) and the competitive ELISA method measures:
10 μ g/ml VEGF165 (NaHCO
3PH9.6) 4 ℃ of wrapper sheets spend the night. and the scFv of serial dilution mixes with ABL-1mAb respectively, hatch (37 ℃ with VEGF165,2h). usefulness HRP link coupled goat anti-mouse igg detection scFv inhibition ABL-1mAb combines with VEGF165's. and result such as Fig. 5, result confirm that ABL-1scFv and ABL-1 monoclonal antibody are that same epi-position combines VEGF165.
3) Western blotting measures:
Get VEGF165 20 μ l, the 13%SDS-PAGE electrophoresis, be transferred to cellulose nitrate film, after the 1%BSA sealing, add scFv and hatch 2h for 37 ℃, anti-His monoclonal antibody (Novagen, USA) be one anti-, HRP link coupled goat anti-mouse igg is two anti-, the TMB colour developing. (specifically with reference to Current protocol inimmunology unit 8.10). and result such as Fig. 6, result show that scFv and monoclonal antibody discern the linear epitope of hsVEGF165.
Sequence table
＜110〉woods value China
＜120〉gene of human vascular endothelial growth factor monoclonal antibody and application thereof
<160>4
<210>1
<211>342
<212>DNA
＜213〉hybridoma cell strain
<220>
<221>
<222>(1)…(342)
<400>1
<210>2
<211>114
<212>PRT
<400>2
<210>3
<211>318
<212>DNA
＜213〉hybridoma cell strain
<400>3
<210>4
<211>106
<212>PRT
<400>4
Claims (10)
1. the gene of human vascular endothelial growth factor monoclonal antibody, it is characterized in that: this gene comprises heavy chain variable region gene and chain variable region gene, heavy chain variable region gene has the nucleotide sequence of SEQ ID NO:1, and chain variable region gene has the nucleotide sequence of SEQ ID NO:3.
2. by the polypeptide of the genes encoding of claim 1, it is characterized in that: this polypeptide has the aminoacid sequence of SEQ ID NO:2 and the aminoacid sequence of SEQ ID NO:4.
3. expression vector is characterized in that: this expression vector contains the chain variable region gene of the nucleotide sequence of the heavy chain variable region gene of nucleotide sequence of SEQ ID NO:1 and SEQ ID NO:3.
4. a kind of expression vector according to claim 3 is characterized in that: this expression vector is pET28a-scFv.
5. the polypeptide expressed according to the expression vector of claim 3 or 4.
6. polypeptide according to claim 5 is used for the application of the detection reagent of the relevant tumour of VEGF165 in preparation.
7. the production method of polypeptide according to claim 5, it is characterized in that comprising the steps: to isolate the heavy chain variable region gene VH and the chain variable region gene VL of antibody from the strain of VEGF165 monoclonal cell, and assembling scFv, scFv with assembling makes up prokaryotic expression carrier, transformed into escherichia coli, make up genetic engineering bacterium, and abduction delivering, to expression product separate, purifying and renaturation.
8. the production method of polypeptide according to claim 7 is characterized in that comprising the steps:
1. from the variable region gene of VEGF165 monoclonal cell strain separation antibody:
Collect well-grown, the hybridoma cell strain of energy secretion of VEGF 165 monoclonal antibodies extracts total RNA,
The design degenerate primer, heavy chain variable region gene VH employing primer VH Back (5 '-AGGTSMARCTGCAGSAGTCWGG-3 ') and primer VH For (5 '-TGAGGAGACGGTGACCGTGGTCCCTTGGCCCCAG-3 '),
Chain variable region gene VL employing primer VL back (5 '-GACATTGAGCTCACCCAGTCTCCA-3 ') and primer VL For (5 '-GTTAGATCTCGAGCTTGGTCCC-3 '),
According to single stage method RT-PCR test kit increase respectively heavy chain variable region gene VH and chain variable region gene VL, adopt the RT-PCR method respectively, angle and get antibody variable region heavy chain variable region gene VH and chain variable region gene VL, the clone enters carrier pMD18-T respectively;
2. heavy chain variable region gene VH, the clone of chain variable region gene VL amplified production:
Design of primers is as follows:
VH P
1(5 '-ACCATG
GAATTCCTGCAGGAGTCTGGTGGCTTG-3 '), contain the EcoRI restriction enzyme site,
VH?P
2(5’-AGAGCCACCTCCGCCTGAACCGCCTCCACCTGAGGAGACGGTG-3’)，
VL?P
1(5’-CAGGCGGAGGTGGCTCTGGCGGTAGTGACATTGAGCTCAC-3’)，
VL P
2(5 '-GTGGTG
AAGCTTTCACTCGAGCTTGGTACCACCTC-3 '), contain the HindIII restriction enzyme site;
Adopt Over-lap PCR, one section flexible linker (G of design between heavy chain variable region gene VH and chain variable region gene VL
4S)
3, VH-linker-VL clones in efficient prokaryotic expression carrier pET28a;
3. efficiently express, purifying, renaturation scFv:
PET28a-scFv transforms and enters BL21 (DE3), and the IPTG abduction delivering is collected the inclusion body precipitation, urea dissolving, Ni
2+-IDA His-bind affinitive layer purification albumen, dilution method and dialysis method are to the target protein renaturation, the albumen of purifying is diluted to renaturation buffer I, renaturation 18～30h, the renaturation buffer II that then albumen packed into, dialysis renaturation 10～15h, ultrafiltration pipe Centricon30 protein concentrate, 13%SDS-PAGE detects, and protein-20 ° C preserves.
9. the production method of polypeptide according to claim 8, it is characterized in that: renaturation buffer I selects 50mMTris-HCl, 0.15mM NaCl, 2M urea, 0.5Mm reduced glutathion, 0.1mM Sleep-promoting factor B, pH 8.0 for use, final concentration is 100 μ g/ml, renaturation 24h.
10. the production method of polypeptide according to claim 8, it is characterized in that: renaturation buffer II selects 50mmol/L Tris-HCl, 0.15mol/L NaCl, pH 8.0 for use.
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| CNA2008101204301A CN101487005A (en) | 2008-09-04 | 2008-09-04 | Gene of human vascular endothelial growth factor monoclonal antibody and use thereof |
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|---|---|---|---|
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011023130A1 (en) * | 2009-08-28 | 2011-03-03 | 江苏先声药物研究有限公司 | Anti-vegf monoclonal antibody and pharmaceutical composition comprising said antibody |
| CN102485753A (en) * | 2010-12-03 | 2012-06-06 | 上海杰隆生物工程股份有限公司 | Human derived heavy chain variable region possessing human vascular endothelial growth factor binding activity |
| CN103012588A (en) * | 2011-09-23 | 2013-04-03 | 武汉吉天朋生物科技发展有限公司 | Targeted therapy of tumors by using McAb capable of neutralizing VEGFA biological activity |
| WO2013170779A1 (en) * | 2012-05-17 | 2013-11-21 | 江苏先声药业有限公司 | Pharmaceutical composition comprising anti-vegf antibody |
-
2008
- 2008-09-04 CN CNA2008101204301A patent/CN101487005A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011023130A1 (en) * | 2009-08-28 | 2011-03-03 | 江苏先声药物研究有限公司 | Anti-vegf monoclonal antibody and pharmaceutical composition comprising said antibody |
| US8986692B2 (en) | 2009-08-28 | 2015-03-24 | Jiangsu Simcere Pharmaceutical R & D Co., Ltd. | Anti-VEGF monoclonal antibody and pharmaceutical composition comprising said antibody |
| CN102485753A (en) * | 2010-12-03 | 2012-06-06 | 上海杰隆生物工程股份有限公司 | Human derived heavy chain variable region possessing human vascular endothelial growth factor binding activity |
| CN103012588A (en) * | 2011-09-23 | 2013-04-03 | 武汉吉天朋生物科技发展有限公司 | Targeted therapy of tumors by using McAb capable of neutralizing VEGFA biological activity |
| WO2013170779A1 (en) * | 2012-05-17 | 2013-11-21 | 江苏先声药业有限公司 | Pharmaceutical composition comprising anti-vegf antibody |
| CN103417965A (en) * | 2012-05-17 | 2013-12-04 | 江苏先声药物研究有限公司 | Anti-VEGF antibody containing pharmaceutical composition |
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