CN102093479B

CN102093479B - Recombinant chimeric antibody of anti-human vascular endothelium growth factor receptor 2

Info

Publication number: CN102093479B
Application number: CN201010564660A
Authority: CN
Inventors: 侯宗柳; 孟明耀; 刘运洪; 朱晓云; 庞伟; 魏传钰; 解燕华
Original assignee: Individual
Current assignee: Individual
Priority date: 2010-11-29
Filing date: 2010-11-29
Publication date: 2012-10-03
Anticipated expiration: 2030-11-29
Also published as: CN102093479A

Abstract

The invention relates to a recombinant chimeric antibody of an anti-human vascular endothelium growth factor receptor 2, a preparation method and application thereof. The preparation method comprises the following steps of: respectively amplifying variable area genes of light chains and heavy chains of antibodies in hamster hybridoma cells, and carrying out chimeric recombination on the variable area genes of the light chains and the heavy chains respectively with genes of constant areas of light chains and heavy chains of human IgG1; constructing the chimeric light chains and the heavy chains of the antibodies into a retrovirus recombinant vector pMSCV-antiVEGFR2-IgG; transfecting Chinese hamster ovary (CHO) cancer cells; and screening and preparing a recombinant chimeric IgG antibody of the anti-human vascular endothelium growth factor receptor 2, wherein the hamster hybridoma cell line D3-D3 is preserved in the China Center for Type Culture Collection (CCTCC) on August 5th, 2010, and the preservation number is CCTCC NO: C201076; and the chimeric IgG antibody resisting VEGFR-2 relates to diagnostic reagents resisting VEGFR-2 and antibody target drugs of tumor vessel suppressive treatment.

Description

The recombined chimeric antibody of human vessel endothelium growth factor resisting acceptor 2

Technical field

The present invention relates to immunological technique field, particularly chimeric antibody and uses thereof, a kind of specifically recombined chimeric antibody of human vessel endothelium growth factor resisting acceptor 2.

Background technology

Immune in recent years targeted therapies demonstrates prospect preferably in clinic diagnosis; Vasculogenesis and relevant vascular diseases (comprising tumour) mainly rely on the regulation and control of angiogenesis factor (VEGF) and angiostatin; Wherein the closest is VEGF and acceptor (VEGFR), particularly VEGF and acceptor 2 (VEGFR-2) thereof.If ideal targeted drug identification VEGR/VEGFR passage is arranged, then can play the effect of diagnosis and treatment disease.

Because the high degree of specificity of antibody, utilizing antibody is a kind of ideal approach with the drug targeting target cell, and current monoclonal antibody drug or antibody coupling medicine have become and increased the fastest, one of the maximum market of making a profit in the pharmaceutical industry.

The antibody engineering technology that is the main body with cell engineering and genetic engineering technique is the research focus in this year at preparation therapeutic antibodies medicine, and it has great potential and application prospect in infection, cardiovascular disorder, autoimmune disorder, oncotherapy.The selectivity of antibody drug action target spot, the humanization of antibody drug, miniaturized and high efficiency also are research emphasis from now on.

Because the reaction of the generation HAMA of the clinical application of mouse resource monoclonal antibody (HAMA) and anaphylaxis; Lowered the curative effect of medicine, thereby the utilization genetic engineering technique is transformed mouse source antibody reduction HAMA reaction; Still the specificity and the avidity that have kept simultaneously antibody; Utilize eukaryotic expression system to express the IgG whole antibody, make the target antibody structure, have more use value more near natural.

Summary of the invention

One of the object of the invention is to provide a kind of recombined chimeric antibody and encoding sox thereof of human vessel endothelium growth factor resisting acceptor 2.

The recombined chimeric antibody of a kind of human vessel endothelium growth factor resisting acceptor 2 of the present invention comprises:

(1) chimeric light chain of the κ constant region of light chain of the variable region of light chain of human vessel endothelium growth factor resisting acceptor 2 mouse source antibody and human normal immunoglobulin IgG composition; Described chimeric light chain contains the aminoacid sequence shown in the SEQ NO1, and SEQ NO1 aminoacid sequence is following: SEQ NO1

PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

And

(2) chimeric heavy chain of the CH of the variable region of heavy chain of human vessel endothelium growth factor resisting acceptor 2 mouse source antibody and human normal immunoglobulin IgG1 composition; Described chimeric heavy chain contains the aminoacid sequence that SEQ NO2 lists, and SEQ NO2 aminoacid sequence is following: SEQ NO2

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

In SEQ NO1 and the SEQ NO2 sequence, following stroke of two horizontal lines partly are leader peptide sequences, and following stroke wavy line partly is a variable region sequences, and uncrossed part is the constant region sequence.

Described chimeric light chain contains the aminoacid sequence that SEQ NO1 lists.The DNA of coding SEQ NO1 aminoacid sequence is following: SEQ NO1 DNA

CCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

Described chimeric heavy chain contains the aminoacid sequence that SEQ NO2 lists.The DNA of coding SEQ NO2 aminoacid sequence is following: SEQ NO2DNA

GCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

In above-mentioned listed SEQ NO1DNA and the SEQ NO2DNA sequence, wherein following stroke of two horizontal lines partly are leader peptide sequences, and following stroke wavy line partly is a variable region sequences, is terminator sequence in the square frame, and uncrossed part is the constant region sequence.

The primer that the present invention uses an Analysis of Nested Design has cloned light chain, heavy chain variable region gene VL and the VH of human VEGFR-3 resistant-2 antibody from the human VEGFR-3 resistant-2 mouse monoclonal antibody mAb hybridoma cell strain of autonomous structure and cultivation; Consistent with corresponding gene height in the conserved regions of biosoftware BLAST analysis revealed VL and VH and the DB, the confirmation inventor has correctly cloned light chain, the heavy chain variable region gene of human VEGFR-3 resistant-2 antibody from the hybridoma cell strain that can secrete mAb through round pcr.The primer that the present invention uses an Analysis of Nested Design again extracts human IgG1's light chain CL and heavy chain CH gene from volunteer's periphery lymphocyte; Consistent with corresponding gene height in biosoftware BLAST analysis revealed CL and CH conserved regions and the DB, the confirmation inventor has correctly cloned human IgG1's light chain CL and heavy chain CH gene from volunteer's periphery lymphocyte through round pcr.The primer that the present invention uses an Analysis of Nested Design again with mouse VL and VH respectively with human IgG1's CL and the chimeric reorganization of CH; Light chain of antibody after chimeric and heavy chain are connected to the front and back that the eukaryotic cell internal ribosome inserts site (IRES); Be connected between CMV promotor and the SV40Poly A; Finally be built into the retrovirus recombinant vectors, transfection CHO cell, screening and the chimeric IgG antibody of preparation anti-VEGFR-2.Test used anti-VEGFR-2 antibody hybridoma cell strain D3-D3 and be preserved in Chinese typical culture collection center on August 5th, 2010, deposit number is CCTCC C201076.

The DNA expression vector that comprises SEQ NO1 and SEQ NO2 sequence is like pIRES, pMSCV or genetically engineered other expression vectors commonly used.

Described dna vector transformed host cells is like Chinese hamster ovary celI or genetically engineered other express cells commonly used.

Two of the object of the invention is to provide the method for a kind of structure and chimeric antibody expression.

Light chain of antibody after chimeric and heavy chain are connected to the front and back that the eukaryotic cell internal ribosome inserts site IRES; Be connected between CMV promotor and the SV40Poly A; The structure form is CMV promotor-chimeric light chain-IRES-chimeric heavy chain-SV40PolyA; Finally be built into expression vector pIRES-antiVEGFR2-IgG and retrovirus expression vector pMSCV-antiVEGFR2-IgG; Fixed IgG1 constant region gene and corresponding restriction enzyme site are arranged on the described pIRES-antiVEGFR2-IgG, can make up to different antigenic animal-people's chimeric antibodies through the different antibody variable gene of displacement.

In order to realize the stably high expression level of chimeric antibody in eukaryotic cell; The present invention has adopted Chinese hamster ovary celI and the retroviral vector expression system that carries Xin Meisu neo gene; The advantage of this system is: chimeric antibody is incorporated on the Chinese hamster ovary celI karyomit(e) through the retroviral vector transfection together with Xin Meisu neo gene; Having only transfection and integration to go up the neo gene could survive, and can filter out the positive cell clone of stablizing the high expression level foreign gene with Xin Meisu easily.

Three of the object of the invention is to provide a kind of preparation method of recombined chimeric antibody of human vessel endothelium growth factor resisting acceptor 2, may further comprise the steps: cultivate foregoing host cell, reclaim expressed antibody.

Utilize 5 ' RACE reverse transcription technology and polymerase chain reaction; The light chain and the heavy chain variable region gene of antibody increase respectively from the mouse hybridoma of secretion human vessel endothelium growth factor resisting acceptor 2 antibody; Extract the light chain of human normal immunoglobulin IgG1 and the gene of CH again; Utilize overlapping polymerase chain reaction Overlap technology with mouse light chain and heavy chain variable region gene respectively with the chimeric reorganization of gene of human IgG1's light chain and CH; The light chain and the heavy chain of chimeric back antibody are connected to the front and back that the eukaryotic cell internal ribosome inserts site IRES; Be connected between cytomegalovirus promoter PCMV and the adenovirus aggregate gland thuja acid SV40Poly A; Finally be built into retrovirus recombinant vectors pMSCV-antiVEGFR2-IgG, transfection Chinese hamster ovary cancer cells CHO, the chimeric IgG antibody of reorganization of screening and preparation human vessel endothelium growth factor resisting acceptor 2; The mouse hybridoma strain D3-D3 of said secretion human vessel endothelium growth factor resisting acceptor 2 antibody is preserved in Chinese typical culture collection center on August 5th, 2010, and deposit number is CCTCC C201076.

To contain correct recombinant retrovirus pMSCV-antiVEGFR2-IgG plasmid liposome transfection GP2-293 cell; 37 ℃ of absorption 6h; Add α-MEM substratum; Get supernatant when cultivating 24h, use final concentration to be the NaCl of 0.45M and 8% the PEG60004 ℃ of deposition of spending the night, obtain recombinant retrovirus pMSCV-antiVEGFR2-IgG with infectivity.

Recombinant retrovirus pMSCV-antiVEGFR2-IgG is infected through expressing the Chinese hamster ovary celI of pressure screening; Serum-free culture; Contain the chimeric IgG antibody of excretory human VEGFR-3 resistant 2 people mouse in 37 ℃ of culture supernatant, collect culture supernatant, cross the SPA affinity column with the speed of 2～10ml/min; Use 5mol/L Guanidinium hydrochloride wash-out then, subsequent use with the PBS dialysis back of 0.001mol/mL again.

Four of the object of the invention is to provide and contains the drug regimen medicine that foregoing recombined chimeric antibody is an activeconstituents, and said composition contains acceptable carrier on described recombined chimeric antibody and the pharmacology.

Said chimeric antibody be used for that preparation suppresses or with VEGF-2 acceptor 2 active medicines; Said chimeric antibody is used for preparing the application of the antibody target medicine treatment of diagnostic reagent and tumor vessel suppression therapy.

The present invention has made up the eukaryon expression plasmid of chimeric IgG antibody and has carried out solubility expression; Set up the purification process of expressing protein; Obtained highly purified soluble proteins; Antibody character and specificity that this albumen has kept mouse source property anti-VEGFR-2 antibody to possess have the potential application prospect at the target diagnosis and treatment medicine that is used for relevant vascular diseases (tumour that comprises some high expression level VEGFR-2).

The advantage that the present invention compared with prior art has:

Fast development along with modern biotechnology; Modern biochemistry and Protocols in Molecular Biologies such as language function genomics, proteomics, information biology; In conjunction with technology such as genetically engineered, protein engineering, cell engineerings, make the biotech drug research and development get into the epoch of developing by leaps and bounds.Therapeutic antibodies is the medicine of the antibody engineering technology preparation that is the main body with cell engineering and genetic engineering technique, with its high specificity, safe and effective and become the focus of current biotech drug research and development.The chimeric IgG antibody of anti-VEGFR-2 people mouse of preparation, the antibody skeleton uses human IgG, and antigen binding site uses VL and the VH of mouse anti VEGFR-2, the chimeric whole antibody structure that is built into, and use eukaryotic system and express, it is natural that the antibody conformation is approached; Show antigenic property and the specificity that has kept mouse source property anti-VEGFR-2 antibody through test; But have stronger specificity and long transformation period than natural antibody; Be used for relevant vascular diseases; The tumor research of high expression level VEGFR-2 particularly can be used for preparing diagnostic reagent, the curative drug of anti-VEGFR-2.

Use recombinant gene that this mouse resource monoclonal antibody is transformed the chimeric IgG antibody of preparation, the antibody fragment of preparation has following advantage:

(1) greatly reduces the mouse derived component of anti-VEGFR-2 antibody, help further carrying out in vivo tests.

(2) keep antibody character and specificity that the mouse endogenous antibody possesses, can potentially be used for the target diagnosis and treatment of relevant vascular diseases (tumour that comprises some high expression level VEGFR-2).

(3) the chimeric whole antibody of eukaryotic expression makes activity stronger more near natural structure, and the transformation period is longer, but great expression is convenient to purifying, as therapeutic antibodies.

Description of drawings

Fig. 1: anti-VEGFR-2 authentic monoclonal antibody cell total rna The results in electrophoresis: show that the RNA extracting is better, do not degrade.

Fig. 2: back monoclonal anti VEGFR-2 antibody chain variable region (mVL) and variable region of heavy chain (mVH) (containing the part constant region) pcr amplification product The results in electrophoresis: M purify: nucleic acid standard molecular weight (Takara DL2000); The about 750bp of mVL (containing the part constant region); The about 750bp of mVH (containing the part constant region).

Fig. 3: with healthy subjects lymphocyte RNA is the human IgG1's of template amplification constant region of light chain (hIgG1CL κ) and CH (hIgG1CH) fragment The results in electrophoresis: M: nucleic acid standard molecular weight (Takara DL2000); The about 350bp of hIgG1CL κ; The about 1000bp of hIgG1CH; Fig. 3 a: constant region of light chain (hIgG1CL κ); Fig. 3 b: CH (hIgG1CH).

Fig. 4: chimeric light chain and heavy chain amplified production 1% agarose gel electrophoresis experimental result: M: nucleic acid standard molecular weight (TakaraDL2000); Fig. 4 a: the about 350bp of variable region of light chain (mVL), the about 350bp of constant region of light chain (CL κ), the about 350bp of chimeric back light chain (LL); Fig. 4 b: the about 350bp of variable region of heavy chain (mVH), the about 1000bp of CH (hIgG1CH), the about 1500bp of chimeric back heavy chain (HH).

Fig. 5: the nucleic acid electrophoresis experimental result of chimeric IgG amplified production: M1: nucleic acid standard molecular weight (Takara DL5000); M2: nucleic acid standard molecular weight (Takara DL15000); 1 for the IgG gene fragment that contains IRES of about 4000bp.

Fig. 6: Western-bloting detects human VEGFR-3 resistant-2 chimeric antibody character and molecular weight experimental result: 1 is human VEGFR-3 resistant-2 chimeric antibody of 50 μ g/ml; 2 is human VEGFR-3 resistant-2 chimeric antibody of 25 μ g/ml; Fig. 6 a is a HRP mark goat anti-human igg antibody detected result, and Fig. 6 b is HRP mark sheep anti mouse Fab antibody test result.

Fig. 7: on HUVEC and AML cell, carry out the immunohistochemical experiment result: wherein one anti-and only add two anti-ly as negative control not add, Fig. 7 a, Fig. 7 b are the negative control of HUVEC and AML cell; Fig. 7 c, Fig. 7 d are respectively the figure that combines of human VEGFR-3 resistant 2 chimeric antibodies and HUVEC and AML cell.

Fig. 8: on HUVEC and AML cell, be at war with and suppress the immunohistochemical experiment result: wherein only to add the negative contrast of recombinant human VEGF, suppress antibody with human VEGFR-3 resistant 2 chimeric antibodies as competition, Fig. 8 a, Fig. 8 b are the negative control of HUVEC and AML cell; Fig. 8 c, Fig. 8 d are that human VEGFR-3 resistant 2 chimeric antibodies combine figure with the competition of people VEGF on HUVEC and AML cell.

Fig. 9: with commercialization recombinant human VEGF R-2 albumen is antigen, and human VEGFR-3 resistant 2 chimeric antibodies are an anti-indirect method ELISA experimental result: wherein a, b are people VEGFR2 chimeric antibody sample, the positive contrast of posi, the negative contrast of neg.

Figure 10: with commercialization recombinant human VEGF R-2 albumen is antigen, the ELISA experimental result that human VEGFR-3 resistant 2 chimeric antibodies and VEGF competition suppress: the positive contrast of Posi, the negative contrast of Neg.Wherein a, b are anti-VEGFR-2 chimeric antibody sample, and 1/2a, 1/2b are the human VEGFR-3 resistant 2 chimeric antibody samples of 1/2 concentration, posi positive control, the negative contrast of neg.

Embodiment

The preparation method of the recombined chimeric antibody of human vessel endothelium growth factor resisting acceptor 2 of the present invention, this antibody fragment is to utilize the genetic engineering technique preparation, concrete steps are following:

(1) segmental amplification of murine antibody variable region gene and sequential analysis

Mouse source property anti-VEGFR-2 antibody hybridoma D3/D5 is cultured to logarithmic phase, Trizol-chloroform-isopropanol method extracting cell total rna; Use SMARTer ^TMRACE cDNA Amplification Kit, wherein the GSPs of mouse variable region of light chain, variable region of heavy chain (gene-specific primers) is respectively:

mCLκB：5-ACACTCATTCCTGTTGAAGCTCTTGACAATGG，

MIgMCH1:5-ACATGCAGATCTCTGTTTTTGCCTCCGTAGTGG is a template with the mRNA among total RNA, and 5 ' RACE reverse transcription amplification obtains to contain the dna segment of variable region of light chain, variable region of heavy chain; The rt condition be 42 ℃ 90 seconds; 70 ℃ 10 minutes; PCR uses KOD plus taq Polyerase system, amplification condition be 95 ℃ 2 minutes; 95 ℃ 20 seconds, 56 ℃ 10 seconds, 70 ℃ 1 minute, 30 circulations; 70 ℃ were extended 5 minutes, and agarose gel electrophoresis all amplifies the band of about 750bp; Glue reclaims purifying amplification purpose band; Add " A " tail, TA is cloned into pMD19T-Simple (TakaRa) carrier then, will connect product and transform the XL1-Blue competence bacteria; Coating contains the LB flat board of 100 μ g/ml penbritins, puts 37 ℃ of 15h; At random the single bacterium colony of picking totally 10 spend the night and shake bacterium, extract plasmid then, serve the Hai Shenggong order-checking; Sequencing result shows through the DNAStar software analysis: VL clone except that have 1 undergo mutation; Other VL sequences are identical, VH clone except that have 3 undergo mutation, other VH sequences are also identical; Identical sequence is confirmed as aim sequence, as follows:

Variable region of light chain VL comprises part constant region: 336bp

GAC ATTGTGATGTCACAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAAGGTCACTAT GAGCTGCAAATCCAGTCAGAGTCTGCTCAACAGTAGAACCCGAAAGAACTACTTGGCT TGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTACTGGGCATCCACTAG GGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCA CCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGCAAGCAATCTTATAATC TGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCA...

Variable region of heavy chain (VH) (comprising the part constant region): 366bp

TAACT

GA GGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGAGAAGCCTGGCGCTTCAGTGAAGATA TCCTGCAAGGCTTCTGGTTACTCATTCACTGGCTACAACATGAACTGGGTGAAGCAGAG CAATGGAAAGAGCCTTGAGTGGATTGGAAATATTGATCCTTACTATGGTGGTACTAGCTA CAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAATCCTCCAGCACAGCC TACATGCAGCTCAAGAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGATCT GGGTATGGTTACGGGGAGGGTTACTACTTTGACTACTGGGGCCAAGGCACCACTCTCAC AGTCTCCTCAGAGAGTC

Sequence is imported the BLAST comparative analysis, and confirm that following stroke of two horizontal line flags sequence are leader sequence: light chain is 60bp, and heavy chain is 336bp, and the sequence of the horizontal line mark that places an order is the antibody variable region sequence: light chain is 60bp, and heavy chain is 366bp.

(2) amplification of human IgG antibody's constant region gene fragment and sequential analysis

Extract the RNA in healthy volunteer's peripheral blood lymphocyte, RT-PCR obtains cDNA, is template with cDNA, and PCR uses KOD plus polyerase system, the human IgG antibody's that increases respectively CL κ and CH chain nucleotide fragments; The upstream primer of CL κ section is hCL κ F:5-T TCTAGACGAACTGTGGCTGCACCATCTGTCTT, downstream primer are hCL κ B:5-T ACGCGTTCACTAACACTCTCCCCTGTTGAAGCTCTTTGTGACG; Wherein the upstream primer of CL κ section is introduced the XbaI restriction enzyme site, and the downstream primer of CL κ section is introduced the MluI restriction enzyme site, and the upstream primer of CH is hCHF:5-AGC GTCGACCAAGGGCCCATCGGTCTTCC, downstream primer are hCHB:5-T GCGGCCGCCTATCATTTACCCGGAGACAGGGAGAGGCTCTTC; The CH upstream primer is introduced the SalI restriction enzyme site, and the CH downstream primer is introduced the NotI restriction enzyme site, amplification condition be 95 ℃ 2 minutes; 95 ℃ 20 seconds, 56 ℃ 10 seconds, 70 ℃ 1 minute, 30 circulations; 70 ℃ were extended 5 minutes, and 1% agarose gel electrophoresis amplifies the band of about 750bp of CL κ and the about 1000bp of CH; Glue reclaims purifying amplification purpose band; Add " A " tail, TA is cloned into pMD19T-Simple (TaKaRa) carrier then, will connect product and transform the XL1-Blue competence bacteria; Coating contains the LB flat board of 100 μ g/ml penbritins, puts 37 ℃ of 15h; 5 of the single bacterium colonies of picking spend the night and shake bacterium at random; Extract plasmid then, serve the Hai Shenggong order-checking, sequencing result and pubmed go up sequence alignment; Show through DNA Star software analysis: each clone of CL κ removes all has point mutation; One of them is numbered, and CL κ #2 clone's aminoacid sequence and reference sequences is identical, and each clone of CH removes to have all has point mutation, and it is identical more than 95% with the IgG1 reference sequences that one of them is numbered aminoacid sequence that CH#5 clones.Identical sequence is confirmed as aim sequence, as follows (sequence of following horizontal line mark is the antibody restriction enzyme site):

The constant region of light chain nucleotides sequence is classified as: 327bp

TCTAGACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

ACGCGT......

The constant region of light chain aminoacid sequence is: 107aa

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC-

CH nucleotide sequence: 996bp

GC GTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

CGGCCGC......

The CH nucleotides sequence is classified as: 330aa

(3) light chain and heavy chain is chimeric

1, light chain is chimeric:

According to the chimeric amplimer of restriction enzyme site light chain in pIRES carrier characteristics and the Overlap PCR principle design band, as follows:

LL1：5-AA GCTAGC

ATGGATTCACAGGCCCAGGTTC

LL2：5-CAGATGGTGCAGCCACAGTTCG TTTGATTTCCAGCTTGGTGCCTC

LL3：5- GAGGCACCAAGCTGGAAATCAAACGAACTGTGGCTGCACCATCTG

LL4：5-TCG ACGCGTCCTGCAGG

ACACTCTCCCCTGTTGAAGC

Primer LL1, LL2 amplification VL fragment wherein; Primer LL3, LL4 amplification CL κ fragment; Introducing restriction enzyme site NheI at 5 of primer LL1 ' end is that the Kozak sequence that underscore partial sequence and eukaryotic expression are used is the square frame partial sequence; Introduced restriction enzyme site MluI and SbfI is that the terminator sequence that underscore partial sequence and eukaryotic expression are used is the square frame partial sequence at 5 of primer LL4 ' end; That uses the underscore mark among primer LL3 and the LL4 is VL afterbody sequence, does not use the unlabelled head end sequence as CL κ of underscore, is beneficial to the VL of mouse source property anti-VEGFR-2 antibody and the segmental overlapping pcr amplification of CL κ of human IgG; The step of amplification: PCR uses KOD plus polyerase system, is template with the VL sequence, with primer LL1, LL2 amplification, obtains product A.With CL κ sequence is template, with primer LL3, LL4 amplification, obtains product B.Agarose gel electrophoresis, product A, B are the band of about 350bp.Product A, B are respectively got balanced mix, and 10 times of redilution as template, advance amplification with primer LL1, LL4, obtain product C, agarose gel electrophoresis, and product C is the band of about 700bp, product C is light chain (CL κ) gene of required chimeric antibody.Above is handled in follow-up TA clone and plasmid extraction etc., and sequential analysis chimeric antibody light chain (CL κ) gene order conforms to design fully.Order-checking institute calling sequence is following:

Chimeric antibody light chain nucleotides sequence is classified as: (663bp; Following stroke of two horizontal line parts is homing sequence in the sequence, and following stroke of single horizontal line partly is restriction enzyme site, and drawing wavy line partly is variable region sequences; Square frame inside is divided into terminator sequence, and uncrossed part is the constant region sequence)

GCTAGC

CCTGCAGGACGCGT

2, heavy chain is chimeric:

HH1：5-A TCTAGA ATGGGATGGACCTGGATC

HH2：5-GATGGGCCCTTGGTCGACG CTGAGGAGACTGTGAGAGTGGTG

HH3：5- CACCACTCTCACAGTCTCCTCAGCGTCGACCAAGGGCCCATC

HH4：5-T GCGGCCGC

TTTACCCGGAGACAGGGAGAGGCTCTTC

Primer HH 1, HH 2 amplification VH fragments wherein; Primer HH 3, HH 4 amplification CH fragments; Having introduced restriction enzyme site XbaI at 5 of primer HH 1 ' end is that the Kozak sequence that underscore partial sequence and eukaryotic expression are used is the square frame partial sequence; Having introduced restriction enzyme site Not at 5 of primer HH 4 ' end is that the terminator sequence that underscore partial sequence and eukaryotic expression are used is the square frame partial sequence; That uses the underscore mark among primer HH 3 and the HH 4 is VH afterbody sequence, does not use the head end sequence as CH of underscore mark, is beneficial to the VH of mouse source property anti-VEGFR-2 antibody and the segmental overlapping pcr amplification of CH of human IgG; The step of amplification: PCR uses KOD plus polyerase system, is template with the VH sequence, with primer HH 1, HH 2 amplifications, obtains product E.With the CH sequence is template, with primer HH 3, HH 4 amplifications, obtains product F.Agarose gel electrophoresis, product E is the band of about 1000bp for band, the F of about 350bp.Product E, F are respectively got balanced mix, and 10 times of redilution as template, advance amplification with primer HH 1, HH4, obtain product G, agarose gel electrophoresis, and product G is the band of about 1500bp, product G is heavy chain (CH) gene of required chimeric antibody.The same steps of treatment process (1) such as follow-up TA clone and plasmid extraction, sequential analysis chimeric antibody light chain (CH) gene order conforms to design fully.Order-checking institute calling sequence is following: the chimeric antibody heavy chain nucleotide sequence is: (1362bp; Following stroke of two horizontal line parts is homing sequence in the sequence; Under draw single horizontal line partly for restriction enzyme site; Drawing wavy line partly is variable region sequences, and square frame inside is divided into terminator sequence, and uncrossed part is the constant region sequence)

TCTAGA

CGGCCGC

(4) chimeric IgG1 Expressed by Retrovirus Vector and evaluation

1, the structure of pIRES-IgG recombinant vectors

Obtain and be connected to plasmid pIRES MCS A with restriction endonuclease NheI and MluI from chimeric light chain (CL κ) gene order T carrier, obtain and be connected to plasmid pIRES MCS B from chimeric heavy chain (CH) gene order T carrier with restriction endonuclease XhoI and NotI.At this moment, light chain and heavy chain place under the control of same promotor CMV promotor, connect a RES sequence (IRES) behind the light chain gene, are heavy chain genes thereafter, and the bicistronic mRNA of transcribing out can be because of the existence of IRES is translated gently simultaneously, heavy chain.

2, pMSCV-antiVEGFR2-IgG retroviral vector construct

The PCR method will be obtained on the reorganization pIRES-IgG carrier from the CMV promotor to the SV40PolyA fragment, and used PCR primer is: IRES1:5-GAC GAATTCTCAATATTGGCCATTAGCCATATTA, IRES2:5-CTT CTCGAGTTTTACCACATTTGTAGAGGTTTTAC, and be connected to pMSCV carrier MCS place through XhoI and EcoRI, obtain the pMSCV-antiVEGFR2-IgG recombinant vectors.Identify definite sudden change of not having through order-checking.

(5) express the Screening and Identification of chimeric IgG antibody cell and the purifying of expressing protein

1, retroviral packing

To contain correct recombinant retrovirus pMSCV-antiVEGFR2-IgG plasmid liposome transfection GP2-293 cell, cultivate 24 holes, put 37 ℃ of 6h after; Add α-MEM substratum; When cultivating 24h, get supernatant and use final concentration to be the NaCl of 0.45M and 8% the PEG60004 ℃ of deposition of spending the night, 4 ℃ of centrifugal 30min of 10000rpm then; Get total RNA of deposition and extraction transfectional cell, detect transfection efficiency with light chain and heavy chain primer PCR.

With the cell precipitation results, after the ultrasonication, frozen in-80 ℃ together with the culture supernatant deposition.

2, human VEGFR-3 resistant-2 chimeric antibody is in the expression of Chinese hamster ovary celI

With frozen pMSCV-antiVEGFR2-IgG, 100CCID in-80 ℃ ₅₀/ ml37 ℃ is adsorbed in Chinese hamster ovary celI 1h, adds serum free medium then, 37 ℃, 5%CO ₂Cultivate, got supernatant in 2-6 days and detect, detect with ELISA method and immunohistochemical methods.

3, human VEGFR-3 resistant 2 chimeric antibody purifying

The culture supernatant of serum-free culture CHO is collected, cross the SPA affinity column, use 5mol/L Guanidinium hydrochloride wash-out then with the speed of 2～10ml/min, subsequent use with 0.001mol/mLPBS dialysis back again.

(6) Function Identification of human VEGFR-3 resistant 2 chimeric antibodies

1, Western-bloting detects human VEGFR-3 resistant-2 chimeric antibody characteristic and molecular weight

Human VEGFR-3 resistant-2 chimeric antibody of human VEGFR-3 resistant-2 chimeric antibody of 50 μ g/ml of eukaryotic expression and 25 μ g/ml is carried out the 4-10%SDS-PAGE electrophoresis and electricity forwards on the nitrocellulose filter; Respectively with HRP mark goat anti-human igg antibody and 4 ℃ of incubated overnight of HRP mark sheep anti mouse Fab antibody; The DAB colour developing; All there is band to show 150kDa place and HRP mark goat anti-human igg antibody and HRP mark sheep anti mouse Fab antibody; Conform to theory, the variable region of containing purpose antibody mouse and people's constant region are described.

2, indirect elisa method detects human VEGFR-3 resistant-2 chimeric antibody avidity

With containing the 0.1M carbonate buffer solution, the coating buffer of pH9.6 dilution recombinant human VEGF R-2 albumen encapsulates 96 hole elisa plates with the concentration of 1 μ g/ml, and every hole adds 100 μ l, and 4 ℃ are spent the night; After the PBST washing 5 times,, hatch 2h for 37 ℃ with the PBS damping fluid sealing that contains 3%BSA; PBST washing 5 times, each 3min; Add 100 μ l human VEGFR-3 resistant-2 chimeric antibodies in two holes, 37 ℃ of 2h, PBST washing 5 times, each 3min, 1h is hatched for 37 ℃ in the HRP mark goat anti-human igg antibody 100 μ l/ holes of using dilute at 1: 5000 again; PBST washing 5 times; Each 3min, TMB colour developing liquid 100 μ l/ holes, room temperature after following 15 minutes with 2M sulfuric acid stopped reaction; Detect and adopt dual wavelength 450nm/630nm, inosculating antibody human VEGFR-3-2 inosculating antibody physical efficiency and recombinant human VEGF R-2 albumen play antigen-antibody ELISA reaction as a result.

3, VEGF and VEGFR2 competition suppresses ELISA

With containing the 0.1M carbonate buffer solution, the coating buffer of pH9.6 dilution recombinant human VEGF R-2 albumen encapsulates 96 hole elisa plates with the concentration of 1 μ g/ml, and every hole adds 100 μ l, and 4 ℃ are spent the night; After the PEST washing 5 times,, hatch 2h for 37 ℃ with the PBS damping fluid sealing that contains 3%BSA; After the PBST washing 5 times, add the chimeric IgG antibody of 100ng/50 μ l anti-VEGFR-2+100ng/50 μ l recombinant human VEGF in each hole, hatch 2h for 37 ℃; After the PBST washing 5 times, add 100ng/100 μ l goat-anti people VEGF antibody and hatch 1h for 37 ℃; PBST washing 5 times, each 3min adds the HRP mark mouse-anti sheep IgG antibody 100 μ l/ holes of diluting at 1: 5000 again, hatches 1h for 37 ℃; PBST washing 5 times; Each 3min, TMB colour developing liquid 100 μ l/ holes, room temperature after following 15 minutes with 2M sulfuric acid stopped reaction; Detect and adopt dual wavelength 450nm/630nm; Do positive control with VEGF100ng/50 μ l+0.002M PBS/50 μ l/ hole in the experiment, 0.002M PBS/10 μ l/ does negative control in the hole, and the result shows that human VEGFR-3 resistant-2 inosculating antibody physical efficiency and recombinant human VEGF protein competition are incorporated into and encapsulates the albumen in the recombinant human VEGF R-2 of 96 orifice plates.

4, immunohistochemical methods

With certain density HUVEC and the film-making of AML cell, dry up the back with 4% formaldehyde fixed 10min, drip the chimeric IgG antibody of 100ng/10 μ l anti-VEGFR-2 (diluent is the PBS of 1%BSA) then; The PBS of 1%BSA makes 4 ℃ of incubated overnight of negative control; Wash 5 times with PBST then, drip HRP mark mouse-anti human IgG antibody, incubated at room 2h; Observe in microscopically, the result shows that human VEGFR-3 resistant-2 inosculating antibody physical efficiency combines with VEGFR2 on the HUVEC cell.

5 and competition suppress immunohistochemical methods

With certain density HUVEC and the film-making of AML cell; Dry up the back with 4% formaldehyde fixed 10min; The chimeric IgG antibody of 100ng/10 μ l anti-VEGFR-2,100ng/10 μ lVEGF, PBS are incorporated into cell sheet, the chimeric IgG antibody of 100ng/10 μ l anti-VEGFR-2+100ng/10 μ lVEGF respectively then; 4 ℃ of incubated overnight are washed 5 times with PBST then; The chimeric IgG antibody of 100ng/10 μ l anti-VEGFR-2 adds HRP mark mouse-anti human IgG antibody;, VEGF, PBS, anti-VEGFR-2 be chimeric+VEGF all adds the antibody of the anti-VEGF of HRP mark; Incubated at room 2h; Observe in microscopically, the result shows that human VEGFR-3 resistant-2 inosculating antibody physical efficiency is competed on the HUVEC cell with VEGF and combines with VEGFR2.

Claims

1. the recombined chimeric antibody of human vessel endothelium growth factor resisting acceptor 2 is characterized in that: comprise

(1) chimeric light chain of the κ constant region of light chain of the variable region of light chain of human vessel endothelium growth factor resisting acceptor 2 mouse source antibody and human normal immunoglobulin IgG composition, the aminoacid sequence of described chimeric light chain is shown in SEQ NO1, and SEQ NO1 aminoacid sequence is following:

And

(2) chimeric heavy chain of the CH of the variable region of heavy chain of human vessel endothelium growth factor resisting acceptor 2 mouse source antibody and human normal immunoglobulin IgG1 composition, the aminoacid sequence of described chimeric heavy chain is shown in SEQ NO2, and SEQ NO2 aminoacid sequence is following:

2. preparation method of the recombined chimeric antibody of human vessel endothelium growth factor resisting acceptor 2 according to claim 1; Comprise and utilize 5 ' RACE reverse transcription technology and polymerase chain reaction; The light chain and the heavy chain variable region gene of antibody increase respectively from the mouse hybridoma of secretion human vessel endothelium growth factor resisting acceptor 2 antibody; Extract the light chain of human normal immunoglobulin IgG1 and the gene of CH again; Utilize overlapping polymerase chain reaction Overlap technology with mouse light chain and heavy chain variable region gene respectively with the chimeric reorganization of gene of light chain and the CH of human IgG l; The light chain and the heavy chain of chimeric back antibody are connected to the front and back that the eukaryotic cell internal ribosome inserts site IRES; Be connected between cytomegalovirus promoter and the adenovirus aggregate gland thuja acid SV40PolyA; Finally be built into retrovirus recombinant vectors pMSCV-antiVEGFR2-IgG, transfection Chinese hamster ovary cancer cells CHO screens and prepares the chimeric IgG antibody of reorganization of anti-human vessel endothelium growth factor resisting acceptor 2; In preservation on August 5 in 2010 to Chinese typical culture collection center, deposit number is CCTCC C201076 to the mouse hybridoma strain D3-D3 of said secretion human vessel endothelium growth factor resisting acceptor 2 antibody.

3. the purposes of the recombined chimeric antibody of the said human vessel endothelium growth factor resisting acceptor 2 of claim 1 is characterized in that: be used for preparation inhibition or and VEGF-2 acceptor 2 active medicines.

4. pharmaceutical composition, it is characterized in that: said composition contains acceptable carrier on recombined chimeric antibody as claimed in claim 1 and the pharmacology.