CN103145843B - Single chain antibody of anti-fibroblast growth factor receptor (anti-FGFR) - Google Patents
Single chain antibody of anti-fibroblast growth factor receptor (anti-FGFR) Download PDFInfo
- Publication number
- CN103145843B CN103145843B CN201310054935.3A CN201310054935A CN103145843B CN 103145843 B CN103145843 B CN 103145843B CN 201310054935 A CN201310054935 A CN 201310054935A CN 103145843 B CN103145843 B CN 103145843B
- Authority
- CN
- China
- Prior art keywords
- chain antibody
- yeast
- antibody
- variable region
- single chain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 229940126864 fibroblast growth factor Drugs 0.000 title claims description 15
- 102000009465 Growth Factor Receptors Human genes 0.000 title 1
- 108010009202 Growth Factor Receptors Proteins 0.000 title 1
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 17
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 5
- 239000002773 nucleotide Substances 0.000 claims description 8
- 125000003729 nucleotide group Chemical group 0.000 claims description 8
- 239000013612 plasmid Substances 0.000 abstract description 48
- 240000004808 Saccharomyces cerevisiae Species 0.000 abstract description 40
- 102100023600 Fibroblast growth factor receptor 2 Human genes 0.000 abstract description 11
- 101710182389 Fibroblast growth factor receptor 2 Proteins 0.000 abstract description 11
- 238000006243 chemical reaction Methods 0.000 abstract description 6
- 239000003814 drug Substances 0.000 abstract description 5
- 239000013604 expression vector Substances 0.000 abstract description 4
- 102000004169 proteins and genes Human genes 0.000 abstract description 4
- 210000003527 eukaryotic cell Anatomy 0.000 abstract description 3
- 210000005253 yeast cell Anatomy 0.000 abstract description 3
- 238000001086 yeast two-hybrid system Methods 0.000 abstract description 3
- 230000003993 interaction Effects 0.000 abstract description 2
- 102000037865 fusion proteins Human genes 0.000 abstract 2
- 108020001507 fusion proteins Proteins 0.000 abstract 2
- 229940079593 drug Drugs 0.000 abstract 1
- 230000006916 protein interaction Effects 0.000 abstract 1
- 238000011084 recovery Methods 0.000 abstract 1
- 239000007788 liquid Substances 0.000 description 39
- 241000894006 Bacteria Species 0.000 description 32
- 239000000243 solution Substances 0.000 description 28
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 18
- 150000001413 amino acids Chemical class 0.000 description 18
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 16
- 239000008103 glucose Substances 0.000 description 16
- 239000006228 supernatant Substances 0.000 description 16
- 235000001014 amino acid Nutrition 0.000 description 15
- 210000004027 cell Anatomy 0.000 description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 15
- 108020004414 DNA Proteins 0.000 description 14
- 206010028980 Neoplasm Diseases 0.000 description 14
- 229910009891 LiAc Inorganic materials 0.000 description 12
- 239000000427 antigen Substances 0.000 description 12
- 108091007433 antigens Proteins 0.000 description 12
- 102000036639 antigens Human genes 0.000 description 12
- 235000015097 nutrients Nutrition 0.000 description 12
- 241000588724 Escherichia coli Species 0.000 description 10
- 230000001954 sterilising effect Effects 0.000 description 10
- 239000000725 suspension Substances 0.000 description 10
- 239000008367 deionised water Substances 0.000 description 9
- 229910021641 deionized water Inorganic materials 0.000 description 9
- 239000000203 mixture Substances 0.000 description 9
- 239000011780 sodium chloride Substances 0.000 description 9
- 238000012360 testing method Methods 0.000 description 9
- 238000010790 dilution Methods 0.000 description 8
- 239000012895 dilution Substances 0.000 description 8
- 239000000843 powder Substances 0.000 description 7
- 239000013589 supplement Substances 0.000 description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 6
- 201000011510 cancer Diseases 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 210000000130 stem cell Anatomy 0.000 description 5
- 101150009006 HIS3 gene Proteins 0.000 description 4
- 102100024594 Histone-lysine N-methyltransferase PRDM16 Human genes 0.000 description 4
- 101000686942 Homo sapiens Histone-lysine N-methyltransferase PRDM16 Proteins 0.000 description 4
- 239000001888 Peptone Substances 0.000 description 4
- 108010080698 Peptones Proteins 0.000 description 4
- 101100394989 Rhodopseudomonas palustris (strain ATCC BAA-98 / CGA009) hisI gene Proteins 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 235000019319 peptone Nutrition 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 239000012154 double-distilled water Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 229920001223 polyethylene glycol Polymers 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- MKPCNMXYTMQZBE-UHFFFAOYSA-N 7h-purin-6-amine;sulfuric acid;dihydrate Chemical compound O.O.OS(O)(=O)=O.NC1=NC=NC2=C1NC=N2.NC1=NC=NC2=C1NC=N2 MKPCNMXYTMQZBE-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 108050007372 Fibroblast Growth Factor Proteins 0.000 description 2
- 102000018233 Fibroblast Growth Factor Human genes 0.000 description 2
- 241000628997 Flos Species 0.000 description 2
- 108091007491 NSP3 Papain-like protease domains Proteins 0.000 description 2
- 108020002230 Pancreatic Ribonuclease Proteins 0.000 description 2
- 102000005891 Pancreatic ribonuclease Human genes 0.000 description 2
- 229920002562 Polyethylene Glycol 3350 Polymers 0.000 description 2
- 108700008625 Reporter Genes Proteins 0.000 description 2
- 239000007984 Tris EDTA buffer Substances 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 230000009849 deactivation Effects 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 230000009182 swimming Effects 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- OPIFSICVWOWJMJ-YGEXGZRRSA-N 5-bromo-4-chloro-3-indolyl alpha-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 OPIFSICVWOWJMJ-YGEXGZRRSA-N 0.000 description 1
- 101150096273 ADE2 gene Proteins 0.000 description 1
- 101710112752 Cytotoxin Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 101150021185 FGF gene Proteins 0.000 description 1
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 description 1
- 102000052575 Proto-Oncogene Human genes 0.000 description 1
- 108700020978 Proto-Oncogene Proteins 0.000 description 1
- 102000004278 Receptor Protein-Tyrosine Kinases Human genes 0.000 description 1
- 108090000873 Receptor Protein-Tyrosine Kinases Proteins 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 108010008038 Synthetic Vaccines Proteins 0.000 description 1
- 102000044209 Tumor Suppressor Genes Human genes 0.000 description 1
- 108700025716 Tumor Suppressor Genes Proteins 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- DWVANBHPEPSMOV-LSCFUAHRSA-N [(2r,3s,4r,5r)-5-[6-(benzylamino)purin-9-yl]-3,4-dihydroxyoxolan-2-yl]methyl dihydrogen phosphate Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(O)=O)O[C@H]1N1C2=NC=NC(NCC=3C=CC=CC=3)=C2N=C1 DWVANBHPEPSMOV-LSCFUAHRSA-N 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 229940125644 antibody drug Drugs 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000002458 cell surface marker Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 231100000599 cytotoxic agent Toxicity 0.000 description 1
- 239000002619 cytotoxin Substances 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 238000012407 engineering method Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000012447 hatching Effects 0.000 description 1
- 102000057593 human F8 Human genes 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- XIXADJRWDQXREU-UHFFFAOYSA-M lithium acetate Chemical compound [Li+].CC([O-])=O XIXADJRWDQXREU-UHFFFAOYSA-M 0.000 description 1
- 108010056929 lyticase Proteins 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 229940047431 recombinate Drugs 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical class OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a single chain antibody of anti-FGFR. The single chain antibody comprises a heavy chain variable region having an amino acid sequence represented by SEQ ID NO.1 and a light chain variable region having an amino acid sequence represented by SEQ ID NO.2. The single chain antibody having an interaction with FGFR2 is screened from a human single chain antibody library by treating the FGFR2 as a target protein through utilizing a yeast two-hybrid system, so yeast cells allow fusion proteins to be expressed in the natural environments of eukaryotic cells as eukaryotic cells. The yeast conversion is convenient and is easy to operate, the conversion efficiency is high, and the plasmid recovery is convenient. The high expression of the fusion proteins is ensured through using a high-copy and strong-promoter expression vector, so the interaction between proteins can be sensitively detected, thereby the single chain antibody having strong specificity is obtained. The screened antibody is completely humanized and can be directly used for exploiting antibody medicines for the human.
Description
Technical field
The invention belongs to antibody field, particularly the single-chain antibody of an anti-fibroblast growth factor acceptor.
Background technology
Malignant tumour, self-discovery just becomes one of great illness threatening human life later always.In order to overcome this global great difficult problem, various countries scientist is working hard.In recent years, utilize specific antibody target cancer stem cell surface marker to become a research direction for the treatment of cancer, find the specific marker thing at all kinds of cancer stem cells surface high expression level, and prepare the committed step that becomes this class research about their antibody of high specific.
The tumor stem cell hypothesis proposing has in recent years been overturned the traditional view about tumour occurrence cause.The universally recognized tumour of people be based on inherent genetic material and the interactional result of external environment.In the process of Growth of Cells, because genetic material is undergone mutation, may cause the activation of proto-oncogene, the unconventionality expression of the reticent and relevant apoptogene of cancer suppressor gene, thus cause the growth of cell no longer controlled, thus tumour generated.Traditional view thinks that such variation is to come from the sudden change that tumor tissues all cells accumulates always, and found by scientific research personnel's research in recent years, in tumor tissues, only have sub-fraction cell to have and form and maintain growth of tumour cell and heterogeneous ability, such sub-fraction cell is referred to as tumor stem cell.Fibroblast growth factor acceptor 2 (FGFR2) is a member in tyrosine kinase receptor, interacts with fibroblast growth factor (FGFs), and the process such as propagation, differentiation of regulating cell.And in kinds cancer cell and their cancer stem cell, having the example of FGFR2 unconventionality expression, the target spot using it as cancer therapy, has researching value.
Single-chain antibody (scFv) is a kind of novel genetically engineered fusion antibody, and it is made up of the variable region of heavy chain of complete antibody and variable region of light chain and the short pliable and tough peptide chain (10～20 amino acid) that is connected both, molecular mass very little (30kDa).The activity of scFv is the same with other antibody, and biological activity is to be determined by the hypervariable region in light chain of antibody and variable region of heavy chain (CDRs) specific gene order.ScFv, owing to having retained the antigen binding capacity of complete antibody, can utilize its affinity to specific objective albumen, and the carrier that sets it as radionuclide goes tumour to carry out radiodiagnosis and treatment.ScFv, due to self, allows it in treatment application, have some advantages: first this small molecules can enter in solid tumor and its hetero-organization faster; Secondly the speed that it removes in blood is also faster, can reduce the injury of normal tissue.In addition, scFv can, with some other material as cytotoxin albumen, medicine and radionuclide merge or coupling, so just can allow it to relative specific target cell effect.At present, do not see yet the report about anti-FGFR2 single-chain antibody.
Summary of the invention
In order to overcome the shortcoming and deficiency of prior art, the object of the present invention is to provide a kind of single-chain antibody of anti-fibroblast growth factor acceptor.
Object of the present invention is achieved through the following technical solutions: a kind of single-chain antibody of anti-fibroblast growth factor acceptor, comprises variable region of heavy chain and variable region of light chain;
The aminoacid sequence of described variable region of heavy chain is as follows:
QVQLQQWGAGLLKPSETLSLTCAVYGGSFS
GYYWSWIRQPPGKGLEWIG
EINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR；
The aminoacid sequence of described variable region of light chain is as follows:
SYELTQLPSVSVSPGQTARISC
SGDALANQHAYWFQQRPGQAPAAVIY
RD TERPSGIPERFSGSRSGTTVTLTISGVQAGDEADYYC
QSAD；
The nucleotide sequence of the variable region of heavy chain described in coding is as follows:
CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGA；
The nucleotide sequence of the variable region of light chain described in coding is as follows:
TCCTATGAGCTGACACAGCTACCCTCGGTGTCAGTGTCCCCAGGACAGACGGCCAGGATCAGCTGCTCTGGAGATGCATTGGCAAACCAACATGCTTATTGGTTCCAGCAGAGGCCAGGCCAGGCCCCTGCGGCGGTCATAATAGAGACACTGAGAGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAGGTCCGGGACAACAGTCACCTTGACCATCAGTGGAGTCCAGGCGGGAGACGAGGCTGACTATTACTGTCAATCAGCAGAC；
Because the biological activity of scFv is to be determined by the hypervariable region in light chain of antibody and variable region of heavy chain (CDRs) specific gene order, therefore, can recombinate to the nucleotide sequence of the encoding heavy chain chain variable region as above providing and encoded light chain variable region by gene engineering method, obtain dissimilar antibody;
The single-chain antibody of described anti-fibroblast growth factor acceptor, is made up of variable region of heavy chain, connection chain and variable region of light chain, and its aminoacid sequence is as follows:
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGRDPHYDILTGSSFDYWGQGTLVTVSSAAAITSYNVYYTKLLARQSYELTQLPSVSVSPGQTARISCSGDALANQHAYWFQQRPGQAPAAVIYRDTERPSGIPERFSGSRSGTTVTLTISGVQAGDEADYYCQSADIIYMCNSAEGPS；
The nucleotide sequence of the single-chain antibody of the anti-fibroblast growth factor acceptor described in coding is as follows:
CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGAGGCCGCGACCCCCATTACGATATTTTGACTGGTTCCTCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCGGCCGCAATAACTTCGTATAATGTGTACTATACGAAGTTATTGGCGCGCCAGTCCTATGAGCTGACACAGCTACCCTCGGTGTCAGTGTCCCCAGGACAGACGGCCAGGATCAGCTGCTCTGGAGATGCATTGGCAAACCAACATGCTTATTGGTTCCAGCAGAGGCCAGGCCAGGCCCCTGCGGCGGTCATATATAGAGACACTGAGAGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAGGTCCGGGACAACAGTCACCTTGACCATCAGTGGAGTCCAGGCGGGAGACGAGGCTGACTATTACTGTCAATCAGCAGACATTATCTACATGTGCAATTCGGCGGAGGGACCGAGC；
The gene of the described variable region of heavy chain of coding is by 291 based compositions, and 97 amino acid of encode, 2 CDR(complementation determinants are contained in variable region) district: CDR1 5 amino acid of encoding, for
gYYWS; CDR2 16 amino acid of encoding, for
eINHSGSTNYNPSLKS, it is to belong to family 4, the single-chain antibody of Germline34;
The gene of the described variable region of light chain of coding is by 273 based compositions, and 91 amino acid of encode, 3 CDR(complementation determinants are contained in variable region) district: CDR1 11 amino acid of encoding, for
sGDALANQHAY; CDR2 7 amino acid of encoding, for
rDTERPS; CDR3 4 amino acid of encoding, for
qSAD, it is to belong to the kappa3 of family, the single-chain antibody of Germline25;
The single-chain antibody of described anti-fibroblast growth factor acceptor can pass through the nucleotide sequence of gene composite coding FGFR2 single-chain antibody FA28, be cloned in expression vector, expression vector after being recombinated again proceeds to corresponding host cell and expresses, purifying, the single-chain antibody of the anti-fibroblast growth factor acceptor described in obtaining.
The present invention has following advantage and effect with respect to prior art:
The present invention is (antigen) using FGFR2 as target point protein, utilize yeast two-hybrid to screen and the interactional single-chain antibody of FGFR2 from mankind's single-chain antibody library, this has following advantage: yeast cell, as eukaryotic cell, can make fusion rotein express in eukaryotic natural surroundings.In addition yeast conversion facilitate easy to operate, transformation efficiency is high, it is convenient to reclaim plasmid.And by using the expression vector of high copy and strong promoter, guaranteed the high expression level of fusion rotein, thereby thereby the interaction between detection albumen that can be sensitive obtains the strong single-chain antibody of specificity.In addition, the antibody screening is total man source section, can directly be used for the antibody drug that exploit person uses.
Brief description of the drawings
Fig. 1 is pGBKT7-FGFR2 double digestion electrophoresis proof diagram, and wherein: the swimming lane on the left side is DNAMarker, the swimming lane on the right is the sample after using EcoRI and BamHI to pGBKT7-FGFR2 double digestion.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited to this.
Embodiment 1
(1) the synthetic antigen plasmid PGBKT7-FGFR2 that obtains of Nanjing Jin Sirui company gene: first in the time of nucleotide sequence (the FGFR2 sequence after optimization) synthetic as follows, add the sticky end of EcoRI restriction enzyme site at 5' end, add the sticky end of BamHI restriction enzyme site at 3' end; Then synthetic sequence is connected with the linear carrier PGBKT7 obtaining by EcoRI and BamHI double digestion, obtains antigen plasmid PGBKT7-FGFR2;
GCAGAAGACTTCGTATCAGAAAACTCAAATAACAAGAGAGCACCTTACTGGACCAACACAGAAAAGATGGAAAAGAGATTACACGCAGTCCCAGCTGCAAATACCGTTAAGTTTAGATGTCCTGCAGGTGGTAATCCAATGCCTACTATGAGATGGTTGAAAAATGGTAAAGAGTTTAAACAAGAACATAGAATCGGTGGTTACAAAGTTAGAAACCAACACTGGTCCTTGATCATGGAATCCGTTGTACCAAGTGATAAGGGTAACTACACTTGTGTCGTTGAAAACGAATACGGTTCTATTAACCATACATACCACTTAGACGTAGTCGAAAGATCACCACATAGACCTATATTACAAGCCGGTTTGCCAGCCAATGCTTCCACAGTTGTTGGTGGTGACGTCGAATTTGTTTGCAAAGTATATAGTGACGCTCAACCTCATATTCAATGGATAAAGCACGTTGAAAAGAATGGTTCTAAGTATGGTCCAGATGGTTTGCCTTACTTAAAGGTTTTGAAGCATTCAGGTATTAATTCTTCAAACGCTGAAGTATTGGCATTATTCAATGTTACCGAAGCAGACGCCGGTGAATATATCTGCAAGGTTTCTAACTACATTGGTCAAGCTAATCAAAGTGCCTGGTTAACAGTATTGCCAAAACAACAAGCCCCTGGTAGAGAAAAAGAA。
Nanjing Jin Sirui company provides the bacillus coli DH 5 alpha that contains antigen plasmid PGBKT7-FGFR2.
(2) from the bacillus coli DH 5 alpha that contains antigen plasmid PGBKT7-FGFR2, extracting antigen plasmid pGBKT7-FGFR2(operates according to OMEGAPlasmid Mini Kit I specification sheets), specific as follows:
1) bacillus coli DH 5 alpha that contains antigen plasmid PGBKT7-FGFR2 is lined on the LB flat board that contains 20 μ g/ml kantlex, cultivate approximately 16 hours for 37 DEG C.
2) well-grown mono-clonal of picking is inoculated in the liquid LB that 5ml contains 20 μ g/ml kantlex, and in 37 DEG C, 200rpm cultivates approximately 16 hours.
3) get 3ml bacterium liquid in the centrifugal 1min of 10000g, supernatant discarded.
4) add 250 μ l Solution I/RNase A, fully mix.
5) add 250 μ l Solution II, put upside down gently 5 times, under room temperature, leave standstill 2min.
6) add 350 μ l Solution III, put upside down gently 5 times, until there is white floss in solution.
7) the centrifugal 10min of 10000g.
8) take out a collection tube, and by DNA column (
miniprep Column) put into pipe.
9) carefully draw supernatant to DNA column, the centrifugal 1min of 10000g, discards the liquid in collection tube.
10) in DNA column, add 500 μ l Buffer HB, the centrifugal 1min of 10000g, discards the liquid in collection tube.
11) in DNA column, add 700 μ l DNA Wash Buffer, the centrifugal 1min of 10000g, discards the liquid in collection tube.
12) repeating step 11).
13) the DNA column 2min of the centrifugal sky of 10000g.
14) whole DNA column is placed on to 37 DEG C of incubators and leaves standstill 5min.
15) DNA column is transferred to a clean EP pipe, adds the centrifugal 1min of 50 μ l aseptic deionized water 10000g, the solution obtaining in EP pipe is exactly the solution that contains pGBKT7-FGFR2 plasmid.
(3) plasmid pGBKT7-FGFR2 is verified: in the centrifuge tube in ice bath at 1.5ml, add 1 μ lEcoRI, 1 μ l BamHI, 2 μ l K buffer and 0.5 μ g plasmid pGBKT7-FGFR2, add subsequently aseptic ddH2O(aseptic double-distilled water), system is supplied to 20 μ l, in 37 DEG C of water-baths, react 1h through row double digestion.The sample that the sepharose of functional quality volume ratio 1% is cut enzyme afterwards carries out electrophoresis, and electrophoretic voltage is 110V, and the time is 30min, and the electrophoresis result obtaining is carried out Taking Pictures recording on gel imaging instrument, as shown in Figure 1.Visible, plasmid pGBKT7-FGFR2 is object plasmid.
(4) the yeast conversion test kit Yeastmaker of use Clontech company
tMyeast Transformation System2(article No. is 630439) (efficient Lithium Acetate method) carry out the conversion of small-scale yeast plasmid:
1) prepare yeast competent cell
A, by yeast AH109(purchased from Clontech company) line on YPDA flat board, be inverted and cultivate 3～5 days at 30 DEG C, from YPDA flat board, picking yeast mono-clonal (diameter 2～3mm) is inoculated in 5mlYPDA liquid nutrient medium, 30 DEG C, 250rpm shaking table cultivation 8～12h.
B, turn yeast liquid that 5 μ l steps A obtain 30 DEG C, 250rpm shaking table in 100mlYPDA liquid culture and cultivate 16～20h, until OD600 reaches 0.15～0.3.
The centrifugal 5min of C, room temperature 1000g collects bacterium liquid, abandons supernatant, and with the fresh resuspended bacterium piece of YDPA liquid nutrient medium of 100ml, 30 DEG C are continued to cultivate 3～5h, until OD600 reaches 0.4～0.5.
D, bacterium liquid is sub-packed in two aseptic centrifuge tubes of 50ml, the centrifugal 5min of room temperature 1000g collects bacterium liquid, abandon after supernatant and use respectively in 1.5ml1.1 × TE/LiAc(test kit 10 × LiAc and 10 × TEbuffer are provided, the formula of 10ml1.1 × TE/LiAc is: 1.1ml10 × LiAc supplements aseptic double-distilled water to 10ml after adding 1.1ml10 × TE buffer) resuspended.
E, bacterium liquid is transferred to respectively in the aseptic EP pipe of two 1.5ml to the centrifugal 60s of 10000g under room temperature.
F, abandon the resuspended bacterium piece of 1.1xTE/LiAc of using respectively 600 μ l after supernatant, be placed in ice bath, obtain yeast competent cell.
2) bait plasmid transformed yeast competence on a small scale
A, in the aseptic EP pipe of 1.5ml, add 100ng antigen plasmid pGBKT7-FGFR2, in the Yeastmaker Carrier DNA(test kit of 5 μ l deactivations, provide, working concentration be 10 μ g/ μ l), in competent cell prepared by 50 μ l step 1) and 500 μ l PEG/LiAc(test kits, provide 10 × TE LiAc, 10 × TEbuffer and 50%PEG3350, the formula of 10ml PEG/LiAc is: 1ml10 × TE LiAc adds 8ml50%PEG3350 after adding 1ml10 × TE buffer again), hatch 30min in 30 DEG C subsequently, every 10min gently springing EP pipe bacterium liquid is mixed.
B, add 20 μ l DMSO and mix after hatching 30min, 15min are placed in 42 DEG C of water-baths, every 5min gently vortex be that bacterium liquid is even.
The centrifugal 15s of C, 10000g collects yeast, abandons after supernatant and adds in 1ml YPD Plus Liquid Medium(test kit and provide) resuspended, 30 DEG C of shaking tables are cultivated 30min.
The centrifugal 15s of D, 10000g collects yeast, resuspended with 1ml0.9% (w/v) NaCl solution.
E, be coated with by getting 100 μ l dilution bacterium liquid after 100 times of resuspended liquid dilutions the SD/-Trp flat board that diameters are 100mm, be inverted for 30 DEG C and cultivate 3～5 days.The yeast clone that so just can tentatively obtain containing antigen plasmid pGBKT7-FGFR2.
(5) the yeast AH109 self activation that contains antigen plasmid pGBKT7-FGFR2 tentatively obtaining detects
X-α-gal is purchased from Sigma company, article No. is 16555, specification is 25mg, using method is that 25mgx-α-gal pressed powder is dissolved in to 1250 μ l dimethyl formamide (dimethylformamide, DMF), be mixed with the storing solution of 20mg/ml, when coated plate, the storing solution of 20mg/ml used with the working fluid that DMF dilution is 4mg/ml.
The yeast AH109 that contains pGBKT7-FGFR2 tentatively obtaining is coated respectively on the SD/-Trp/-Leu/-His/-Ade flat board that SD/-Trp/-Leu is dull and stereotyped and diameter is 100mm that the SD/-Trp flat board that diameter is 100mm (being coated with x-α-gal of 100 μ l, 4mg/ml on it) and diameter are 100mm, observe ADE2, the expression of HIS3 and MEL1.If wherein yeast can be on the flat board of Ade defect normal growth, illustrate that ADE2 gene expresses, if can be on the flat board of HIS defect normal growth, the gene that HIS3 is described is expressed, if can there is blue colonies on the flat board that contains x-α-gal, illustrate that the gene of MEL1 is expressed.The yeast clone that obtains self activating reporter gene MEL1, HIS3 and ADE2, called after pGBKT7-FGFR2 yeast, for the screening of library plasmid.
(6) utilize yeast-two hybrid technique to screen in single-chain antibody library and FGFR2 can interactional single-chain antibody (utilize the yeast conversion test kit Yeastmaker of Clontech company
tMyeastTransformation System2 transforms library plasmid on a large scale in the yeast AH109 that contains antigen plasmid pGBKT7-FGFR2)
1) the AH109 yeast competent cell preparation that contains pGBKT7-FGFR2 plasmid
The AH109 yeast cell that A, inoculation contain pGBKT7-FGFR2, in SD/-Trp solid medium flat board, is inverted for 30 DEG C and is cultivated approximately 4 days.
B, a well-grown bacterium colony of picking (2～3mm) are inoculated in 5ml SD/-Trp liquid nutrient medium, and 30 DEG C, 200rpm cultivates approximately 12 hours.
C, shift 10 μ l bacterium liquid to the fresh SD/-Trp liquid nutrient medium of 100ml, be positioned over 30 DEG C, 200rpm and cultivate approximately 18 hours, until reach 0.15～0.3 to 0D600 value.
The centrifugal 5min of 1000g under D, room temperature, supernatant discarded, adds the SD/-Trp liquid nutrient medium that 100ml is fresh resuspended, and 30 DEG C, 200rpm cultivates approximately 4 hours, until OD600 reaches 0.4～0.5.
E, its point is filled to two 50ml centrifuge tubes, the centrifugal 5min of 1000g, supernatant discarded.
F, every pipe add 1.5ml1.1 × TE/LiAc resuspended.
G, suspension is transferred to respectively in two EP pipes to centrifugal 15 seconds of 10000g, supernatant discarded.
F, every pipe add 600 μ l1.1 × TE/LiAc resuspended, obtain the AH109 yeast competent cell that contains pGBKT7-FGFR2 plasmid.
2) screening and the interactional antibody of FGFR2 in single-chain antibody library
A, get 15ml pipe, add library plasmid PS50-SCFV(Baiao tai Biological Sci & Tech. (Guangzhou) Co., Ltd.) 10 μ g, in the Yeastmaker Carrier DNA(test kit of deactivation, provide, working concentration is l) 20 μ l of 10 μ g/ μ, the AH109 yeast competent cell that 600 μ l contain pGBKT7-FGFR2 plasmid and 2.5ml PEG/LiAc, mix gently.
B, whole system are hatched 45min at 30 DEG C of incubators, and every 15min mixes once.
C, add 160 μ l DMSO, mix.At 42 DEG C of hot activation 20min, vortex is once gently for every 10min.
The centrifugal 5min of D, 1000g, supernatant discarded, adds 3ml YPD Plus Liquid Medium, and 30 DEG C, 200rpm are cultivated 90min.
The centrifugal 5min of E, 1000g, supernatant discarded, adds the NaCl solution 1ml of mass volume ratio 0.9% resuspended.
The NaCl solution that F, the resuspended liquid that step e is obtained take out 11 μ l and 89 μ l mass volume ratios 0.9% fully mixes, and obtains the bacteria suspension of 1:10 dilution by volume; Then get the bacteria suspension of 10 μ l 1:10 dilution by volume and the NaCl solution of 90 μ l mass volume ratios 0.9% and fully mix, obtain the bacteria suspension of 1:100 dilution by volume; By preceding step, obtain the bacteria suspension of 1:1000 dilution by volume.Respectively the bacteria suspension of 1:10,1:100,1:1000 dilution is by volume coated in the SD/-Trp/-Leu flat board that diameter is 100mm, calculated transformation efficiency.
In G, step e, take out bacterium liquid remaining after 11 μ l bacteria suspensions in 1.34 × 10
4the centrifugal 60s of r/min, after removal supernatant mixes with the NaCl solution that 200 μ l mass volume ratios are 0.9%, coating the diameter that contains SD/-Trp/-Leu/-His/-Ade solid medium is in 150mm culture dish.
H, in 30 DEG C of incubators, cultivate 3～5 days, can tentatively obtain containing can and the yeast clone of the interactional single-chain antibody plasmid of FGFR2.
(7) to sieve positive yeast clone carry out reporter gene MEL1, HIS3 and ADE2 line checking:
The yeast positive colony to be detected being sieved to is lined to the SD/-Trp/-Leu flat board that diameter is the x-α-gal that is coated with 200 μ l, 4mg/ml of 150mm, each flat board divides six region line, being positioned over subsequently 30 DEG C of incubators cultivates 7 days, find that there is the blue clone of change and just continued to line diameter the SD/-Trp/-Leu/-His/-Ade flat board that is coated with 200 μ l, 4mg/ml that is 150mm, each plate divides six region line, be positioned over subsequently 30 DEG C of incubators and cultivate 7 days, observe and whether have blue clone to occur.There is blue clone to occur representing that this clone is positive.
(8) be verified as plasmid vector in positive yeast extracting (according to
yeast Plasmid Kit specification sheets operation)
1) picking is verified as positive yeast mono-clonal and is inoculated in 5ml SD/-Trp, cultivates approximately 20 hours in 30 DEG C, 200rpm.
2) draw 5ml bacterium liquid, the centrifugal 1min of 10000g, supernatant discarded.
3) in resuspended thing, add 480 μ l Buffer SE, 10 μ l beta-mercaptoethanols and 20 μ l Lyticase Solution, re-suspended cell, puts 30 DEG C of water-baths and hatches 45min.
4) whole system is in the centrifugal 1min of 10000g, supernatant discarded.
5) add 250 μ l Buffer YP I/RNase A resuspended, add subsequently 50mg glass pellet, fully whirlpool 5min, shifts suspension to another clean EP pipe.
6) add 250 μ l YP II, put upside down gently 5 times, leave standstill 2min in room temperature.
7) add 350 μ l YP III, put upside down gently 5 times, in solution, occur white floss.By whole system in the centrifugal 10min of 10000g.
8) DNA column is put into the collection tube of 2ml, carefully drawn supernatant in step 7) and add in DNA column, the centrifugal 2min of 10000g, discards the liquid in collection tube.
9) add 500 μ l Buffer HB in DNA column, the centrifugal 1min of 10000g, discards the liquid in collection tube.
10) add in 700 μ l DNAWash Buffer to DNA columns, the centrifugal 1min of 10000g, discards the liquid in collection tube.
11) repeating step 10).
12) the centrifugal empty DNA column 2min of 10000g is to remove alcohol.
13) DNA column is transferred to a clean EP pipe, adds 50 μ l Elution Buffer or aseptic deionized water to cylinder, room temperature leaves standstill 1～2min, the centrifugal 1min of 10000g, and the solution obtaining is exactly extracted plasmid solution.Measure plasmid concentration.
(9) the positive yeast plasmid extracting is transformed in bacillus coli DH 5 alpha (green the skies company) amplification, and (plasmid extracting from positive yeast has two classes, one is antigen plasmid, one is corresponding antibody plasmid, the antibiotics resistance of two plasmids is different, antibody plasmid is ammonia benzyl Amp resistance, thereby can utilize this feature that the plasmid extracting in positive yeast clone is transformed in bacillus coli DH 5 alpha, and it is cultivated on the LB flat board that contains Amp, the antibody plasmid that just only contains Amp resistance in the DH5 α bacterium colony that can grow out like this, the antibody plasmid obtaining is used for turning round and sequencing analysis)
The green skies of concrete grammar reference company single stage method competence bacterium is prepared the specification sheets that test kit provides:
A, the competent preparation of bacillus coli DH 5 alpha:
1) DH5 α glycerol stock (test kit provides) being lined is directly the LB solid plate of 100mm, is positioned over 37 DEG C of incubators and cultivates 16h, treats that mono-clonal bacterium colony is longer;
2) mono-clonal bacterium colony of picking, in 5ml LB liquid nutrient medium, at 37 DEG C, is cultivated 16h in the shaking table of 200rpm;
3) get 500 μ l steps 2) in cultivate the bacterium liquid of 16 hours and be added in the fresh LB liquid nutrient medium of 50ml, in 37 DEG C, the shaking table of 200rpm goes out to cultivate after 2～2.5h, until OD600 reaches 0.3～0.5.
4) cultured bacterium liquid in step 3) is collected to bacterium for centrifugal 5 minutes in 4 DEG C of 2000g.Then the resuspended bacterium of LB liquid nutrient medium (placing the LB liquid nutrient medium of 5min on ice bath) of using 5ml precooling, with adding 5ml competence that reagent (providing in test kit) is provided on ice bath, mixes, and places 5 minutes at ice bath.
5) the bacterium liquid finally obtaining in step 4) is sub-packed in the centrifuge tube of 1.5ml to every pipe packing 100 μ l, the competence of the DH5 α that obtains preparing.
B, yeast plasmid are transformed into bacillus coli DH 5 alpha:
1) the yeast positive colony extracting plasmid according to step (8), step (7) being obtained.Then the DH5 α competence bacterium that the yeast plasmid of getting 10 μ l extractings adds 100 μ l to divide to install is placed 30 minutes on ice bath;
2) subsequently whole system is positioned over to 42 DEG C of water-baths, heat-shocked 2 minutes;
3) after heat-shocked, be placed in immediately ice-water bath, 2 minutes;
4) add subsequently 900 μ l LB liquid nutrient mediums, cultivate 1 hour in 37 DEG C, 200rpm;
5) by cultured bacteria suspension in the centrifugal 1min of 2000g, remove supernatant, then the NaCl solution that is 0.9% with 100 μ l mass volume ratios is resuspended, and 100 μ l suspensions after resuspended are all coated to the LB flat board that contains 100 μ g/ml penbritins that diameter is 100mm.In escherichia coli cloning that can be longer, just only contain antibody plasmid.
(10) clone who line has been verified turns round checking: escherichia coli cloning step (9) being obtained by step (2) carries out plasmid extraction (all changing the condition of 20 μ g/ml kantlex in step 2 into 100 μ g/ml penbritins).Then this plasmid is proceeded in the AH109 yeast competent cell that contains pGBKT7-FGFR2 according to step (4), after resuspended the NaCl solution of transformed bacteria liquid use 1ml plasmid volume ratio 0.9%, by volume dilute 100 times and be applied to the SD/-Trp/-Leu/-His/-Ade flat board that diameter is 100mm (being coated with 100 μ l, 4mg/ml x-α-gal on it) above, put into 30 DEG C of incubators and cultivate 3～5 days observationss.It is blue that the clone who occurs on flat board all can become, and further illustrating be sieved to positive colony is true positives.
(11) extracting plasmid, order-checking: the escherichia coli cloning extracting plasmid (all changing the condition of 20 μ g/ml kantlex in step 2 into 100 μ g/ml penbritins) step (9) being obtained by step (2).Design upstream primer is that 5'-TATACGCGTTGATGGCCCAGCCGGC-3' and downstream primer are 5'-GCTCTAGACTTCTTCTTGGGTGCCATGG-3', and the single-chain antibody sequence on plasmid is carried out to sequencing analysis, as follows:
CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGAGGCCGCGACCCCCATTACGATATTTTGACTGGTTCCTCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCGGCCGCAATAACTTCGTATAATGTGTACTATACGAAGTTATTGGCGCGCCAGTCCTATGAGCTGACACAGCTACCCTCGGTGTCAGTGTCCCCAGGACAGACGGCCAGGATCAGCTGCTCTGGAGATGCATTGGCAAACCAACATGCTTATTGGTTCCAGCAGAGGCCAGGCCAGGCCCCTGCGGCGGTCATATATAGAGACACTGAGAGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAGGTCCGGGACAACAGTCACCTTGACCATCAGTGGAGTCCAGGCGGGAGACGAGGCTGACTATTACTGTCAATCAGCAGACATTATCTACATGTGCAATTCGGCGGAGGGACCGAGC；
The sequence recording is carried out data comparison in the lg of NCBI blast, the essential information that obtains single-chain antibody is as follows: the gene of encoding heavy chain variable region is by 291 based compositions, 97 amino acid of encoding, the complementary determinants of 2 CDR(are contained in variable region) district: CDR1 5 amino acid of encoding, for
gYYWS; CDR2 16 amino acid of encoding, for
eINHSGSTNYNPSLKS, it is to belong to family 4, the single-chain antibody of Germline34; The gene of encoded light chain variable region is by 273 based compositions, and 91 amino acid of encoding, the complementary determinants of 3 CDR(are contained in variable region) district: CDR1 11 amino acid of encoding, for
sGDALANQHAY; CDR2 7 amino acid of encoding, for
rDTERPS; CDR3 4 amino acid of encoding, for
qSAD, it is to belong to the kappa3 of family, the single-chain antibody of Germline25.
In embodiment, the substratum of using is composed as follows:
1. LB liquid nutrient medium: in every liter of substratum,, containing 10g NaCl, 10g peptone and 5g yeast powder, deionized water is settled to 1L.121 DEG C of high pressure steam sterilization 15min.
2. LB solid medium: in every liter of substratum,, containing 10g NaCl, 10g peptone, 5g yeast powder and 20g agar powder, deionized water is settled to 1L.121 DEG C of high pressure steam sterilization 15min.
3. YPDA solid medium: 20g peptone+10g yeast powder+15ml concentration is adenine sulfate+20g agar powder of mass volume ratio 0.2%, deionized water dissolving, regulating pH is that 6.5 rear deionized waters are settled to 950ml, 121 DEG C of autoclaving 15min, be cooled to 55 DEG C of left and right, the final content of glucose solution 50ml(glucose that to add through the concentration of 121 DEG C of sterilizing 15min be mass volume ratio 40% is mass volume ratio 2%).
4. YPDA liquid nutrient medium: 20g peptone+10g yeast powder+15ml concentration is the adenine sulfate of mass volume ratio 0.2%, deionized water dissolving, regulating pH is that 6.5 rear deionized waters are settled to 950ml, 121 DEG C of autoclaving 15min, be cooled to 55 DEG C of left and right, the final content of glucose solution 50ml(glucose that to add through the concentration of 121 DEG C of sterilizing 15min be mass volume ratio 40% is mass volume ratio 2%).
5. SD/-Trp substratum (1L)
In 10 of note: 100ml ×-Trp DO Supplement, contain-Trp DO Supplement amount is 0.74g.
Glucose solution is preparation and sterilizing (121 DEG C, 15min) separately, medicine except glucose adds deionized water 950ml, regulate pH to 6.5,121 DEG C of autoclaving 15min, be cooled to 55 DEG C of left and right, then to add the concentration of sterilizing be that the final content of glucose solution 50ml(glucose of mass volume ratio 40% is 2%).
6. SD/-Trp/-Leu substratum (1L)
In 10 of note: 100ml ×-Trp/-Leu DO Supplement, contain-Trp/-Leu DO Supplement amount is 0.64g.Glucose solution is preparation and sterilizing (121 DEG C, 15min) separately, medicine except glucose adds deionized water 950ml, regulate pH to 6.5,121 DEG C of autoclaving 15min, be cooled to 55 DEG C of left and right, then to add the concentration of sterilizing be that the final content of glucose solution 50ml(glucose of mass volume ratio 40% is 2%).
7. SD/-Trp/-Leu/-His/-Ade substratum (1L)
In 10 of note: 100ml ×-Trp/-Leu/-His/-Ade DO Supplement, contain-Trp/-Leu/-His/-Ade DO Supplement amount is 0.6g.Glucose solution is preparation and sterilizing (121 DEG C, 15min) separately, medicine except glucose adds deionized water 950ml, regulate pH to 6.5,121 DEG C of autoclaving 15min, be cooled to 55 DEG C of left and right, then to add the concentration of sterilizing be that the final content of glucose solution 50ml(glucose of mass volume ratio 40% is 2%).
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
Claims (4)
1. a single-chain antibody for anti-fibroblast growth factor acceptor, is characterized in that comprising variable region of heavy chain and variable region of light chain;
The aminoacid sequence of described variable region of heavy chain is as shown in SEQ ID NO.1;
The aminoacid sequence of described variable region of light chain is as shown in SEQ ID NO.2.
2. the single-chain antibody of anti-fibroblast growth factor acceptor according to claim 1, is characterized in that: the gene order of the variable region of heavy chain described in coding is as shown in SEQ ID NO.3;
The following SEQ ID of the gene order NO.4 of the variable region of light chain described in coding shows.
3. the single-chain antibody of anti-fibroblast growth factor acceptor according to claim 1, is characterized in that being made up of variable region of heavy chain, connection chain and variable region of light chain, and its aminoacid sequence is as shown in SEQ ID NO.5.
4. the nucleotide sequence of the single-chain antibody of coding anti-fibroblast growth factor acceptor claimed in claim 3, is characterized in that: described nucleotide sequence is as shown in SEQ ID NO.6.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310054935.3A CN103145843B (en) | 2013-02-20 | 2013-02-20 | Single chain antibody of anti-fibroblast growth factor receptor (anti-FGFR) |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310054935.3A CN103145843B (en) | 2013-02-20 | 2013-02-20 | Single chain antibody of anti-fibroblast growth factor receptor (anti-FGFR) |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103145843A CN103145843A (en) | 2013-06-12 |
| CN103145843B true CN103145843B (en) | 2014-10-29 |
Family
ID=48544200
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310054935.3A Expired - Fee Related CN103145843B (en) | 2013-02-20 | 2013-02-20 | Single chain antibody of anti-fibroblast growth factor receptor (anti-FGFR) |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103145843B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106279417B (en) * | 2016-08-30 | 2019-09-06 | 上海交通大学 | Target the single-chain antibody and its preparation method and application of JAML receptor |
| CN108517012B (en) * | 2018-04-16 | 2020-04-24 | 温州医科大学 | Preparation of fully human antagonistic antibody with basic fibroblast growth factor receptor 1 as target |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102131524A (en) * | 2008-11-07 | 2011-07-20 | 星系生物科技责任有限公司 | Monoclonal antibodies to fibroblast growth factor receptor 2 |
| CN102905723A (en) * | 2010-05-11 | 2013-01-30 | Aveo制药公司 | Anti-FGFR2 antibodies |
-
2013
- 2013-02-20 CN CN201310054935.3A patent/CN103145843B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102131524A (en) * | 2008-11-07 | 2011-07-20 | 星系生物科技责任有限公司 | Monoclonal antibodies to fibroblast growth factor receptor 2 |
| CN102905723A (en) * | 2010-05-11 | 2013-01-30 | Aveo制药公司 | Anti-FGFR2 antibodies |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103145843A (en) | 2013-06-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107814845B (en) | Novel anti-PD-1 nano antibody and application thereof | |
| CN110981958B (en) | PD-L1 antibody | |
| CN104804093A (en) | Single-domain antibody for CD47 | |
| CN103864927B (en) | Retinol conjugated protein (RBP) single domain antibody encoding sequence and application thereof | |
| CN112552402A (en) | Anti-human EGFR nano antibody and application | |
| CN103421115A (en) | CD38 nanometer antibody and application | |
| CN106831981A (en) | A kind of single domain antibody peptide backbone and preparation method thereof | |
| CN106674349A (en) | Improved anti-VEGFR (vascular endothelial growth factor receptor)-2 monoclonal antibody | |
| CN106164268A (en) | The host cell utilizing artificial endosymbiont is modified | |
| CN101891818B (en) | Single-chain antibody in anti-human prostate-specific membrane antigen extracellular region and application thereof | |
| CN103145843B (en) | Single chain antibody of anti-fibroblast growth factor receptor (anti-FGFR) | |
| CN102875674A (en) | Anti-tetanotoxin antibody, and preparation method and application thereof | |
| CN109384851A (en) | The preparation method and applications of third generation LMP2A CAR-T cell | |
| CN103451218B (en) | Preparation method of novel cytokine fusion protein IP10 single-chain antibody | |
| CN102174106B (en) | Anti-Met humanized Fab, anti-Met humanized Fab and doxorubicin conjugate and preparation method and application of anti-Met humanized Fab and doxorubicin conjugate | |
| CN103342750A (en) | Apolipoprotein B nano antibody and coding sequence and application thereof | |
| CN114773484B (en) | Recombinant fusion protein for adsorbing cytokines, nano assembly, and preparation method and application thereof | |
| CN101143902A (en) | Anti-HER2 single-chain antibody-cefuroxime sodium enhanced fusion protein HER2(Fv-LDM) | |
| CN100376690C (en) | Screening method for human gastric cancer drug-resistant cell specific combination and drug-resistant reverse short peptide and sequence | |
| CN101781364A (en) | Interleukin-1 receptor antagonist | |
| CN101921335B (en) | Hybrid tumor generating anti-AMP-18 (Antrum Mucosalprotein-18) monoclonal antibody as well as anti-AMP-18 monoclonal antibody and application thereof in gastric carcinoma detection | |
| CN102617734A (en) | Antibody Dab-2 against FGF-2 and application thereof | |
| CN103408667B (en) | Cystatin C nano antibody and coding sequence thereof | |
| CN102516382A (en) | Antimicrobial peptide Hainanenin-5 of Amolops hainanensis, gene of antimicrobial peptide Hainanenin-5 of Amolops hainanensis, separation and purification method and chemical synthesis method for antimicrobial peptide Hainanenin-5 of Amolops hainanensis and application of gene of antimicrobial peptide Hainanenin-5 of Amolops hainanensis | |
| CN103172721B (en) | Amolops hainanensis antimicrobial peptide Hainanenin-1, and gene, separation purification, chemical synthesis and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20141029 |