CN101029082A - Recombination and preparation of human monocloned antibody against EGFR - Google Patents
Recombination and preparation of human monocloned antibody against EGFR Download PDFInfo
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- CN101029082A CN101029082A CN 200610057985 CN200610057985A CN101029082A CN 101029082 A CN101029082 A CN 101029082A CN 200610057985 CN200610057985 CN 200610057985 CN 200610057985 A CN200610057985 A CN 200610057985A CN 101029082 A CN101029082 A CN 101029082A
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Abstract
A process for preparing the humanized monoclonic antibody of EGFR by recombination is disclosed. Said monoclonic antibody can suppress the growth of human tumor cells expressing EGFR by combining it with the EGFR on the surface of tumor cell to prevent the combination of EGFR and EGF needed by reproduction of tumor cells.
Description
Technical field:
The present invention relates to a kind of recombination and preparation of Humanized monoclonal antibodies, be particularly related to a kind of method of utilizing gene recombination technology to prepare the biologically active polypeptides of human monocloned antibody against EGFR, this monoclonal antibody can be used for suppressing expressing the growth of the human body tumour cell of EGFR, its principle is that this monoclonal antibody combines with the EGFR of the tumor cell surface of high expression level EGFR, combines and the generation effect with the EGFR of cell surface to stop the needed EGF of tumor cell proliferation.
Background technology:
The mitotic the first step of epidermic cell is exactly the membranous type glycoprotein E GF receptors bind of EGF and cell surface and the activation that produces.(Carpenter，et al.，Epidermal Growth Factor，Annual Review Biochem.，Vol.48，193-216(1979))。
Have been found that some class tumour cell high expression level EGFR at present, give expression to the EGF acceptor more much higher than normal cell such as transitional cell bladder carcinoma cell line, breast cancer cell etc.At present with the expression amount of the EGFR sign as patient with breast cancer's prognosis situation, the patient of high expression level EGFR often prognosis is poor.Therefore, high expression level EGFR is thought the risk factor that kinds of tumor cells produces by academia.
EGFR is a member of the ErbB of tyrosine kinase receptor family.EGFR mediates the signal transmission of a plurality of systems that act on the neocytoplasm generation in tumour cell.Tumour cell overexpression EGFR indication can be developed the antagonist of EGFR as the human tumor medicine.
HXE225 is special Humanized monoclonal antibodies at the EGFR1 extracellular region, is used for the treatment of the human tumor of overexpression EGFR1.
This patent provides a kind of preparation method who utilizes hybridoma and high-density suspension culture to efficiently express human monocloned antibody against EGFR HXE225.This patent provides the method for a kind of large-scale industrial production human monocloned antibody against EGFR HXE225, sets up the technology platform of the different monoclonal antibodies of a kind of mass preparation simultaneously.
Summary of the invention:
The invention provides a kind of method for preparing human monocloned antibody against EGFR HXE225, this method may further comprise the steps:
A. the corresponding gene in synthetic HXE225 variable region of heavy chain amino acid coding region;
B. the corresponding gene in synthetic HXE225 variable region of light chain amino acid coding region;
C. the HindIII-NheI site of the gene insertion vector pCMV2 that obtains of step a, pCMV2 contains CMV promotor and human normal immunoglobulin γ 1 constant region encoding gene; The plasmid DNA Transformed E .coli XL-1 that connects, correct through screening and determined dna sequence conclusive evidence clone, recombinant plasmid called after pCMV-ch225-HC;
D. the HindIII-BsiW1 site of the gene insertion vector pGSK that obtains of step b, the pGSK carrier contains CMV promotor and human normal immunoglobulin κ chain constant region and NADPH-linked glutamate synthase encoding gene; The plasmid DNA Transformed E .coli XL-1 that connects, correct through screening and determined dna sequence conclusive evidence clone, recombinant plasmid called after pGS-ch225-LC;
E. the pCMV-ch225-HC plasmid DNA is isolated through the BglII-BamH1 double digestion and contained CMV promotor and human normal immunoglobulin γ 1 constant region encoding gene fragment; The gained fragment is inserted the BamH1 site of pGS-ch225-LC, and Transformed E .coli XL-1 is correct through PCR and DNA plan preface conclusive evidence clone, successfully constructs pGS-ch225-LH.
The f.pGS-ch225-LH recombinant plasmid is by electroporation transfection mouse hybridization bone marrow tumour cell NSO, with selecting substratum at 96 orifice plates screening positive cell clone;
G. screen the cell clone of high expression level with the ELISA method;
H. express the NSO cell subclone of monoclonal antibody HXE225;
The domestication of the high expressing cell strain of i. being screened and extensive suspension culture;
J. monoclonal antibody purification HXE225 and determination of activity;
Wherein the corresponding gene in the described synthetic human keratinocyte Growth Factor of a, b amino acid coding region is a prior art, can obtain by prior art, as the method for synthetic.
PCMV2, pGSK described in step c, the d can make up or buy the commercialization expression vector by Protocols in Molecular Biology.
The DNA sequences encoding of the corresponding gene in the preferred HXE225 variable region of heavy chain of the present invention amino acid coding region is seen sequence table 1 and 3.
The DNA sequences encoding of the corresponding gene in the preferred HXE225 variable region of light chain of the present invention amino acid coding region is seen sequence table 1 and 3.
Preparation method of the present invention is characterized in that, wherein said suspension culture is that the engineering cell strain with qualified reorganization HXE225 is inoculated in the cell cultures jar and cultivates.
Described suspension culture is characterised in that used substratum is a serum-free albumen chemical ingredients substratum, and substratum can be bought commercial substratum (as the CD-Hybridoma medium of Gibco or the serum free medium of JRH) or be prepared according to the principle of cell cultures by manufacturer.
Preparation method of the present invention, wherein said extraction chromatography purification step (1) comprises centrifugal collection supernatant liquor; (2) centrifuged supernatant by ultrafiltration with supernatant concentration; (3) ProteinA affinity chromatography, SP-Sepharose FF, Q-Sepharose FF on the supernatant liquor after will concentrating obtain the HXE225 of 95% above purity.
The present invention is according to the variable region of light chain of HXE225 primary structure, variable region of heavy chain amino acid coding (this sequence can use according to versatility, degeneracy and the genetic code of genetic code preference principle change), synthetic light chain DNA complete sequence.Principal feature of the present invention is, respectively this encoding gene fragment cloning is gone into suitable expression vector, (preferred as: pCMV2, pGSK, can buy from the market or make up voluntarily) the middle formation recombinant plasmid that makes up, then that the recombinant plasmid transfection is suitable cell strain, preferred as: NSO, CHO (can buy from the market), the engineering cell strain that obtains through screening with high expression level ability.This project cell strain obtains highly purified HXE225 through suspension culture after the separation and purification of expressed fusion protein process suitable step.Through analyses such as-terminal amino acid sequencing, mass spectrum molecular weight determination, isoelectric point determination, peptide figure analysis and biological activity assay, the result shows that we expressed HXE225 is in full accord with theory.
The ultimate principle of the molecular cloning method that the present invention uses, and employed raw material in the operation steps, solvent, instrument belongs to the prior art field, as the synthetic method of DNA, and the selection of restriction enzyme site etc., the expression vector that the present invention uses, host bacterium etc. all is commercialization carrier or bacterial classification.
The concrete operations step of above method can be seen embodiments of the invention.
Advantage of the present invention mainly shows and is to efficiently express HXE225, every liter of pure product of HXE225 that nutrient solution can obtain 400mg.
Description of drawings:
Fig. 1 pCMV2 plasmid map and multiple clone site and sequence
Fig. 2 pGSK plasmid map and multiple clone site and sequence
Fig. 3 construction of recombinant plasmid synoptic diagram
Fig. 3-1Construction of pCMV-HXE225 HC
Fig. 3-2Construction of pGS-HXE225 LC
Fig. 3-3Construction of pGS-HXE225 L-H
Fig. 3-4HXE225 expression vector
Fig. 4 HXE225 purge process electrophoretogram
The variable region of light chain dna sequence dna of sequence 1 human monocloned antibody against EGFR HXE225
The light chain variable region amino acid sequence of sequence 2 human monocloned antibody against EGFR HXE225
The light chain dna sequence dna of sequence 3 human monocloned antibody against EGFR HXE225
The light-chain amino acid sequence of sequence 4 human monocloned antibody against EGFR HXE225
The variable region of heavy chain dna sequence dna of sequence 5 human monocloned antibody against EGFR HXE225
The weight chain variable region amino acid sequence of sequence 6 human monocloned antibody against EGFR HXE225
The heavy chain dna sequence dna of sequence 7 human monocloned antibody against EGFR HXE225
The heavy chain amino acid sequence of sequence 8 human monocloned antibody against EGFR HXE225
Embodiment:
Further specify the present invention by the following examples, but not as limitation of the present invention.
The structure and the screening of embodiment 1 engineering cell strain
1. expression vector
Select for use expression vector such as pCMV2, pGSK as expression vector, pCMV2 contains CMV promotor and human normal immunoglobulin γ 1 constant region encoding gene; (see figure 1).
Expression vector pGSK contains CMV promotor and human normal immunoglobulin κ chain constant region and NADPH-linked glutamate synthase encoding gene.(see figure 2)
2, the design of gene and synthetic: according to the primary structure encoding sequence of HXE225, the light chain of synthetic HXE225 and the DNA sequences encoding of variable region of heavy chain.See sequence table 1,2,3,4.
3.HXE225 chain variable region gene inserts the HindIII-NheI site of carrier pCMV2, pCMV2 contains CMV promotor and human normal immunoglobulin γ 1 constant region encoding gene; The plasmid DNA Transformed E .coli XL-1 that connects, correct through screening and determined dna sequence conclusive evidence clone, recombinant plasmid called after pCMV-ch225-HC;
The HXE225 heavy chain variable region gene inserts the HindIII-BsiW1 site of carrier pGSK, and the pGSK carrier contains CMV promotor and human normal immunoglobulin κ chain constant region and NADPH-linked glutamate synthase encoding gene; The plasmid DNA Transformed E .coli XL-1 that connects, correct through screening and determined dna sequence conclusive evidence clone, recombinant plasmid called after pGS-ch225-LC;
The pCMV-ch225-HC plasmid DNA isolated through the BglII-BamH1 double digestion contain CMV promotor and human normal immunoglobulin γ 1 constant region encoding gene fragment; The gained fragment is inserted the BamH1 site of pGS-ch225-LC, and Transformed E .coli XL-1 is correct through PCR and DNA plan preface conclusive evidence clone, successfully constructs pGS-ch225-LH.Make up collection of illustrative plates and see Fig. 3
4. with the recombinant plasmid pGS-ch225-LH transfection NSO cell that successfully constructs, screen the engineering cell strain of high expression level, and the cell strain that is screened is carried out subclone, the final cell strain called after NSO225 of acquisition.
Embodiment 2 suspension culture
1. with the seed cell liquid of adherent culture, be inoculated into the cell cultures jar, substratum is in CD-Hybridoma media or other appropriate media) in cultivate, during stream add and concentrate nutrient solution such as amino acid or glucose etc.; Cell concn reaches 2 * 10
6/ ml, the centrifugal 30min of 14000g collects supernatant liquor, abandons precipitation.
2. the supernatant molecular weight cut-off of Shou Jiing is that the ultra-filtration membrane of 50000D carries out ultrafiltration and concentration.
3. carry out affinity chromatography through Protein A on the spissated supernatant, the chromatography level pad is 20mM PB, pH7.0, and last sample is used the 100mM sodium citrate buffer solution after the overbalance buffer solution elution is not conjugated protein, and the pH3.0 wash-out obtains pure HXE225 just;
4. just pure HXE225 adjusts pH to 5, upward carries out ion exchange chromatography with 20mM PB pH5.0 damping fluid equilibrated SP-Sepharose FF, arrives baseline with 20mM PB pH5.0 buffer solution elution behind the last sample, uses 20mM PB pH7.0,600mM NaCl elution samples;
5.SP-after the ultrafiltration of ion exchange chromatography gained sample damping fluid is changed to 20mM PB pH8.0, arrive baseline with 20mM PBpH8.0 buffer solution elution behind the last sample, use 20mM PB pH8.0 then, 500mM NaCl elution samples.
| OD | Ertibus | 0.2143 | 0.224 | 0.2237 | 0.2043 | 0.1917 | 0.13 | 0.0487 | 0.0117 | 0.0063 | 0.005 |
| OD | Ch225 | 0.2097 | 0.2177 | 0.2247 | 0.214 | 0.1367 | 0.1463 | 0.0837 | 0.0377 | 0.0067 | 0.0057 |
| ％ of initial | Ertibus | 100 | 104.5 | 104.4 | 95.3 | 89.5 | 60.7 | 22.7 | 5.5 | 2.9 | 2.3 |
| ％ of initial | Ch225 | 100 | 103.8 | 107.2 | 102.1 | 65.2 | 69.8 | 39.9 | 18 | 3.2 | 2.7 |
| lg(％ of initial) | Ertibus | 2 | 2.0191 | 2.0187 | 1.9791 | 1.9518 | 1.7832 | 1.356 | 0.7404 | 0.4624 | 0.3617 |
| lg(％ of initial) | Ch225 | 2 | 2.0162 | 2.0162 | 2.009 | 1.8142 | 1.8439 | 1.601 | 1.2553 | 0.5051 | 0.4314 |
| Concentration(M/L) | 0 | 0.2 | 0.4 | 0.6 | 0.8 | 1 | 1.2 | 1.4 | 1.6 | 1.8 | |
| OD | Ertibus | 0.2143 | 0.224 | 0.2237 | 0.2043 | 0.1917 | 0.13 | 0.0487 | 0.0117 | 0.0063 | 0.005 |
| OD | Ch225 | 0.2097 | 0.2177 | 0.2247 | 0.214 | 0.1367 | 0.1463 | 0.0837 | 0.0377 | 0.0067 | 0.0057 |
| ％ of initial | Ertibus | 100 | 104.5 | 104.4 | 95.3 | 89.5 | 60.7 | 22.7 | 5.5 | 2.9 | 2.3 |
| ％ of initial | Ch225 | 100 | 103.8 | 107.2 | 102.1 | 65.2 | 69.8 | 39.9 | 18 | 3.2 | 2.7 |
| lg(％ of initial) | Ertibus | 2 | 2.0191 | 2.0187 | 1.9791 | 1.9518 | 1.7832 | 1.356 | 0.7404 | 0.4624 | 0.3617 |
| lg(％ of initial) | Ch225 | 2 | 2.0162 | 2.0162 | 2.009 | 1.8142 | 1.8439 | 1.601 | 1.2553 | 0.5051 | 0.4314 |
| Concentration(M/L) | 0 | 0.2 | 0.4 | 0.6 | 0.8 | 1 | 1.2 | 1.4 | 1.6 | 1.8 | |
Embodiment 3 ELISA measure expression amount
(pH9.6) monoclonal antibody of Xi Shi anti-human IgG is 4 ℃ of overnight incubation, then with the PBS flushing that contains 0.1%Tween-20 three times for 0.01M Na2CO3,0.035M NaHCO3 with the 100ul carbonate buffer solution with the ELISA assay plate; Pipette 100ul cells and supernatant night to measuring the hole from 96 orifice plates, incubated at room 60 minutes with the PBS flushing that contains 0.1%Tween-20 three times, adds the segmental monoclonal antibody of anti-human IgG Fc that coupling has horseradish peroxidase then then; Incubated at room 60 minutes with the PBS flushing that contains 0.1% Tween-20 three times, is measured OD then after the color reaction
405
1. cell A431 inoculates 96 orifice plates with 2 * 104/ holes, and 37 ℃ are spent the night.PBS gives a baby a bath on the third day after its birth inferior.
2. the fixing 10min of acetone 1: 1.PBS washes secondary.
3. add 10ug/ml, every hole 100ul one resists, and 37 ℃, 2h, PBST give a baby a bath on the third day after its birth inferior.Add 0.1MPB respectively, the every hole 100ul of NH4SCN of PH6.0 preparation from 0.5,1,1.5,2,2.5,3,3.5,4,4.5,5,5.5,6,6.5,7,7.5,8M/L, every extent of dilution 2-3/ hole.Room temperature 15min, PBST give a baby a bath on the third day after its birth inferior.
4. add two anti-(mouse anti HIgG Fc-HRP) and add substrate, chromogenic assay.
Concen.(M/L) Ertibus Ch225
0 2 2
0.2 2.0191 2.0162
0.4 2.0187 2.0162
0.6 1.9791 2.009
0.8 1.9518 1.8142
1 1.7832 1.8439
1.2 1.356 1.601
1.4 0.7404 1.2553
1.6 0.4624 0.5051
1.8 0.3617 0.4314
2 0.1139 0.3802
Result: Ch225 1.5M/L, Ertibux 1.4M/L
Embodiment 5 HXE225 antibody fluidic cells are measured
Fluidic cell is measured (FCM) method: the A431 cell recovery is cultivated in 10%NBS DMEM substratum, treated that cell grow when going down to posterity certain number, peptic cell prepares cell suspension, counts.Get 5 * 106 cells and wash 2 times with the PBS of precooling, 1000 left the heart 5 minutes.The BSA that dissolves in PBS that adds 3ml2%, put 4 ℃ 30 minutes.1000 left the heart 5 minutes, washed 2 times with the PBS of precooling, added washing lotion 2ml at every turn.1000 left the heart 5 minutes, added sample 20ug, put 4 ℃ of reactions 1 hour, washed 2 times with the PBS of precooling.The two anti-2ul that add the FITC mark, put 4 ℃ 30 minutes, wash 2 times with the PBS of precooling, 1000 left the heart 5 minutes, added 400ulPBS, FCM detects.
Sequence table
The variable region of light chain dna sequence dna of sequence 1 human monocloned antibody against EGFR HXE225 (1-66 coded signal peptide)
ATGGATTTTCAGGTGCAGATTTTCAGCTTCTTGCTAATCAGTGCCTCAGTTGCAATGTCC 60
AGAGGAGACATCCAGCTGACCCAGTCTCCAGTCATCCTGTCTGTGAGTCCAGGAGAAAGA 120
GTCAGTTTCTCCTGCAGGGCCAGTCAGAGTATTGGCACAAACATACACTGGTATCAGCAA 180
GAGTCTGAAGATATTGCAGATTATTATTGTCAACAAAATAATAACTGGCCAACCACGTTC 360
GGTGCTGGGACCAAGCTGGAGATCAAA 380
Sequence 2
The light chain variable region amino acid sequence of human monocloned antibody against EGFR HXE225 (1-22 is a signal peptide sequence)
5 10 15
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala
20 25 30
Ser Val Ala Met Ser Arg Gly Asp Ile Gln Leu Thr Gln Ser Pro
35 40 45
Val Ile Leu Ser Val Ser Pro Gly Glu Arg Val Ser Phe Ser Cys
50 55 60
Arg Ala Ser Gln Ser Ile Gly Thr Asn Ile His Trp Tyr Gln Gln
65 70 75
Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile Lys Tyr Ala Ser Glu
80 85 90
Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly
95 100 105
Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser Glu Asp Ile
110 115 120
Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr Thr Phe
125
Gly Ala Gly Thr Lys Leu Glu Ile Lys
The light chain dna sequence dna of sequence 3 human monocloned antibody against EGFR HXE225
ATGGATTTTCAGGTGCAGATTTTCAGCTTCTTGCTAATCAGTGCCTCAGTTGCAATGTCC 60
AGAGGAGACATCCAGCTGACCCAGTCTCCAGTCATCCTGTCTGTGAGTCCAGGAGAAAGA 120
GTCAGTTTCTCCTGCAGGGCCAGTCAGAGTATTGGCACAAACATACACTGGTATCAGCAA 180
GAGTCTGAAGATATTGCAGATTATTATTGTCAACAAAATAATAACTGGCCAACCACGTTC 360
GGTGCTGGGACCAAGCTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTC 420
CCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAAC 480
TTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAAC 540
CTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCAT 660
CAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGA 710
The light-chain amino acid sequence of sequence 4 human monocloned antibody against EGFR HXE225
5 10 15
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala
20 25 30
Ser Val Ala Met Ser Arg Gly Asp Ile Gln Leu Thr Gln Ser Pro
35 40 45
Val Ile Leu Ser Val Ser Pro Gly Glu Arg Val Ser Phe Ser Cys
50 55 60
Arg Ala Ser Gln Ser Ile Gly Thr Asn Ile His Trp Tyr Gln Gln
65 70 75
Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile Lys Tyr Ala Ser Glu
80 85 90
Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly
95 100 105
Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser Glu Asp Ile
110 115 120
Ala Asp Tyr Tyr Cys Gln Gln Asn Ash Asn Trp Pro Thr Thr Phe
125 130 135
Gly Ala Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro
140 145 150
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
155 160 165
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
170 175 180
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
185 190 195
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
200 205 210
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
215 220 225
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
230 235
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys ***
Sequence 5
The variable region of heavy chain dna sequence dna of human monocloned antibody against EGFR HXE225 (1-57 coded signal peptide)
ATGGACTTTGGGCTCAGCTTCATTTTCCTTGCCCTTATTTTAAAAGGTGTCCAGTGTCAG 60
GTACAACTGCAGGAGTCAGGACCTGGCCTAGTGCAGCCCTCACAGAGCCTGTCCATCACC 120
TGCACAGTCTCTGGTTTCTCATTAACTAACTATGGTGTACACTGGGTTCGCCAGTCTCCA 180
ATGAACAGTCTGCAATCTAATGACACAGCCATATATTACTGTGCCAGAGCCCTCACCTAC 360
TATGATTACGAGTTTGCTTACTGGGGCCAAGGGACCACGGTCACCGTTTCCTCT 410
The weight chain variable region amino acid sequence of human monocloned antibody against EGFR HXE225 (1-19 is a signal peptide sequence)
5 10 15
Met Asp Phe Gly Leu Ser Phe Ile Phe Leu Ala Leu Ile Leu Lys
20 25 30
Gly Val Gln Cys Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
35 40 45
Val Gln Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly
50 55 60
Phe Ser Leu Thr Asn Tyr Gly Val His Trp Val Arg Gln Ser Pro
65 70 75
Gly Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Ser Gly Gly Asn
80 85 90
Thr Asp Tyr Asn Thr Pro Phe Thr Ser Arg Leu Ser Ile Asn Lys
95 100 105
Asp Asn Ser Lys Ser Gln Val Phe Phe Lys Met Asn Ser Leu Gln
110 115 120
Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala Arg Ala Leu Thr Tyr
125 130 135
Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr
138
Val Ser Ser
The heavy chain dna sequence dna of sequence 7 human monocloned antibody against EGFR HXE225
ATGGACTTTGGGCTCAGCTTCATTTTCCTTGCCCTTATTTTAAAAGGTGTCCAGTGTCAG 60
GTACAACTGCAGGAGTCAGGACCTGGCCTAGTGCAGCCCTCACAGAGCCTGTCCATCACC 120
TGCACAGTCTCTGGTTTCTCATTAACTAACTATGGTGTACACTGGGTTCGCCAGTCTCCA 180
ATGAACAGTCTGCAATCTAATGACACAGCCATATATTACTGTGCCAGAGCCCTCACCTAC 360
TATGATTACGAGTTTGCTTACTGGGGCCAAGGGACCACGGTCACCGTTTCCTCTGCTAGC 420
ACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACA 480
GCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAAC 540
TACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATC 660
TGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGCAGAGCCCAAATCT 720
TGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCA 780
GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTC 840
GACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACG 960
TACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTAC 1020
AAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCC 1080
AAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCTCGGGATGAGCTGACC 1140
GAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGAC 1260
TCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG 1320
GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAG 1380
AGCCTCTCCCTGTCCCCGGGTAAATGA 1400
The heavy chain amino acid sequence of sequence 8 human monocloned antibody against EGFR HXE225
5 10 15
Met Asp Phe Gly Leu Ser Phe Ile Phe Leu Ala Leu Ile Leu Lys
20 25 30
Gly Val Gln Cys Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
35 40 45
Val Gln Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly
50 55 60
Phe Ser Leu Thr Asn Tyr Gly Val His Trp Val Arg Gln Ser Pro
65 70 75
Gly Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Ser Gly Gly Asn
80 85 90
Thr Asp Tyr Asn Thr Pro Phe Thr Ser Arg Leu Ser Ile Asn Lys
95 100 105
Asp Asn Ser Lys Ser Gln Val Phe Phe Lys Met Asn Ser Leu Gln
110 115 120
Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala Arg Ala Leu Thr Tyr
125 130 135
Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr
140 145 150
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
155 160 165
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
170 175 180
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
185 190 195
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
200 205 210
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
215 220 225
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
230 235 240
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser
245 250 255
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
260 265 270
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
275 280 285
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
290 295 300
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
305 310 315
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
320 325 330
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
335 340 345
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
350 355 360
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
365 370 375
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
380 385 390
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
395 400 405
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
410 415 420
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
425 430 435
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
440 445 450
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
455 460 465
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
468
Pro Gly Lys***
Claims (10)
1. recombinate and prepare the method for human monocloned antibody against EGFR HXE225 for one kind; It is characterized in that, but the coding HXE225 monoclonal antibody variable region of light chain of synthetic, the gene fragment clone in heavy chain border area are arrived suitable expression vector establishment recombinant plasmid, with the engineering cell strain of recombinant plasmid transfection host cell construction expression HXE225 monoclonal antibody.
2. according to the method for claim 1, it is characterized in that, be not limited to sequence 1,2 sequences that indicate but the gene in described variable region of heavy chain, light chain border area is preferred.
3. according to the method for claim 1, it is characterized in that described expression vector is preferred but be not limited to pGSK.
4. according to the method for claim 1, it is characterized in that described cell strain is preferred but be not limited to the NSO cell strain.
5. according to the method for claim 1, it is characterized in that described method makes up the complete DNA sequences encoding of light chain and heavy chain for substep.
6. according to the method for claim 1, it is characterized in that, described heavy chain is configured to synthetic variable region of light chain DNA sequences encoding is inserted among the pCMV2, constitutes complete heavy chain encoding sequence HXE225-HC with constant region of light chain on it, and formed plasmid is called pCMV-ch225-HC.
7. according to the method for claim 1, it is characterized in that, described light chain is configured to synthetic variable region of light chain DNA sequences encoding is inserted among the pGSK, constitutes complete heavy chain encoding sequence HXE225-LC with constant region of light chain on it, and formed plasmid is called pGS-ch225-LC.
8. according to the method for claim 1, it is characterized in that described recombinant plasmid successfully constructs pGS-ch225-LH for the appropriate site of the CMV promotor of described HXE225-HC of claim 7 and upstream thereof being inserted the described pGS-ch225-LC of claim 8 after with restriction enzyme cutting, separation and purification.
9. according to the method for claim 1, it is characterized in that described pGS-ch225-LH transfection NSO cell construction is expressed the engineering cell strain of HXE225 monoclonal antibody.
10. the described light chain of claim 2, variable region of heavy chain dna sequence dna are preferred but to be not limited to sequence 1,5 described.
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