CN100345966C - Human acid fibroblast growth factor resistant single-chain antibody gene - Google Patents
Human acid fibroblast growth factor resistant single-chain antibody gene Download PDFInfo
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- CN100345966C CN100345966C CNB2005101190451A CN200510119045A CN100345966C CN 100345966 C CN100345966 C CN 100345966C CN B2005101190451 A CNB2005101190451 A CN B2005101190451A CN 200510119045 A CN200510119045 A CN 200510119045A CN 100345966 C CN100345966 C CN 100345966C
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Abstract
The present invention relates to a single-chain antibody gene for resisting a human acid fibroblast growth factor, a recombination carrier and an expression product of the gene, which belongs to the technical field of gene engineering. The single-chain antibody (single-chainFv, ScFv) of the present invention has practical value. The ScFv is a single peptide chain formed by two variable regions in a head-tail connection mode, the two variable regions are formed by connecting a light chain (VL) and a heavy chain (VH) by a strip of connecting peptide, and the two variable regions are correctly folded to form fragments having the function of antigen combination by non-covalent bonds. The molecular weight of the ScFv is small and is one sixth of the molecular weight of the complete IgG, the ScFv is easy to penetrate into the exterior of solid tissues and tumors and has the characteristic and the function of parent antibody and antigen combination, so the ScFv is an immunotherapy preparation having application value.
Description
Technical field
The invention belongs to gene engineering technology field, relate to single-chain antibody gene and the recombinant vectors and the expression product of human acid fibroblast growth factor resistant specifically.
Background technology
Acid fibroblast growth factor (acid fibroblast growth factor, aFGF) be one of fibroblast growth family member, can influence growth, differentiation and the function of various kinds of cell such as mesenchymal cell, endocrine cell, neurocyte, in normal physiological and pathologic process, participate in growing and the tissue injury repair process.
Studies show that recently the aFGF gene mRNA all is overexpression in transitional cell carcinoma of bladder, ovarian cancer and borderline ovarian tumors tissue, compare that there were significant differences with normal ovarian, ovary tumor-like lesion and benign tumor of ovary.When canceration takes place when, because the effect of carcinogenic factor makes aFGF gene activity enhancing in ovarian cancer tissue's cell, causes synthetic excessive aFGF, and aFGF further causes the formation of vascular endothelial cell and capillary vessel in the ovarian cancer tissue, thereby quickened the transfer of oncocyte with blood.
The biological significance of expressing about aFGF has caused that scholars pay close attention in recent years, people's expectation utilizes the antagonist inhibition aFGF of aFGF and combining as the means that suppress tumor cell proliferation of its acceptor, effectively suppresses the propagation of tumour cell at the monoclonal anti physical efficiency of aFGF.With the monoclonal antibody is the history in existing 20 years of research of the tumor targeting therapy of carrier, mostly that adopts at present is the mouse endogenous antibody, can cause human antimouse antibody (HAMA) when being applied to interior therapeutic, it has disturbed antibody preparation reasonable distribution and effect in vivo.Secondly, because antibody is macromole, the interior pressure of matter is higher than healthy tissues again between tumour, causes antibody or conjugates low excessively in the picked-up of tumour target site.
Genetic engineering antibody is the utilization recombinant DNA technology, and the antagonist gene carries out process and remould and ressembles, through the suitable expressed antibody molecule of recipient cell.The present invention is to provide actual value single-chain antibody (single-chain Fv, ScFv).ScFv is with light chain (VL) and heavy chain (VH) the end to end single peptide chain in formed two variable regions that connects together by a connection peptides, by correct folding, two variable regions are formed the fragment with antigen combined function by non covalent bond, the ScFv molecular weight is little only to be 1/6 of complete IgG,, easily penetrate solid tissue and inside tumor, have the characteristic and the function of parental antibody conjugated antigen, therefore, ScFv is the immunity therapeutic preparation with potential using value.
Mostly treatment in the market is mouse source property with monoclonal antibody drug (also being " biological missile "), can cause human antimouse antibody (HAMA) when being used for human body, and it has disturbed antibody preparation reasonable distribution and effect in vivo.Secondly, because antibody is macromole, the interior pressure of matter is higher than healthy tissues again between tumour, causes antibody or conjugates low excessively in the picked-up of tumour target site.In addition, the method for special cell cultures makes monoclonal antibody be difficult to scale operation, compare with the chemical pharmaceutical factory or the microorganism synthetic drug plant of identical scale, monoclonal antibody drug found the factory and production process in the fund that consumes very big.Simultaneously, monoclonal antibody can be decomposed under the condition of high temperature and strong acid and strong base, must preserve under zero absolute temperature, otherwise will lose activity fully in several weeks; Antibody capable is by the very fast degraded of Digestive tract, thereby stoped it to enter the periphery of brain or some tumours.Numerous disease can't be treated with monoclonal antibody drug, has restricted the clinical application of monoclonal antibody to a great extent.
Summary of the invention
The aminoacid sequence that the purpose of this invention is to provide the anti-people aFGF-ScFv gene of coding and infer thus; The engineering bacteria that contains described anti-people aFGF-ScFv expression carrier and utilize this carrier to transform is provided; The process for preparing recombinant anti human aFGF-ScFv from described engineering bacteria is provided simultaneously.
Recombinant anti human aFGF-ScFv gene of the present invention is VL and the gene VH that is connected antibody by connection peptides, sets up an anti-people aFGF-ScFv gene, and this gene is by 714 based compositions.
The nucleotides sequence of this people aFGF-ScFv gene is classified as:
atg?acc?cag?tct?cca?gca?atc?ctg?tct?gca?tct?cca?ggg?gag?aag?gtc 48
aca?atg?act?tgc?agg?gcc?agc?tca?agt?gta?agt?tac?atg?cac?tgg?tac 96
cag?cag?aag?cca?gga?tcc?tcc?ccc?aaa?ccc?tgg?att?tat?gcc?aca?tcc 144
aac?ctg?gct?tct?gga?gtc?cct?gct?cgc?ttc?agt?ggc?agt?ggg?tct?ggg 192
acc?tct?tac?tct?ctc?aca?atc?agc?aga?gtg?gag?gct?gaa?gat?gct?gcc 240
act?tat?tac?tgc?cag?cag?tgg?agt?agt?aac?cca?ccc?acg?ttc?ggt?gct 288
ggc?acc?aag?ctg?gaa?atc?aaa?cgg?ggt?gga?ggc?ggt?tca?ggc?gga?ggt 336
ggc?tct?ggc?gga?ggt?ggc?tct?cag?gtg?aag?ctg?cag?cag?tca?ggg?gga 384
ggc?tta?gtg?aag?cct?gga?ggg?tcc?ctg?aaa?ctc?tcc?tgt?gca?gcc?tct 432
gga?ttc?gct?ttc?agt?agc?tat?gac?atg?tct?tgg?gtt?cgc?cag?act?ccg 480
gag?aag?agg?ctg?gag?tgg?gtc?gca?acc?att?agt?agt?ggt?ggt?agt?tac 528
acc?tac?tat?cca?gac?agt?gtg?aag?ggc?cga?ttc?acc?atc?tcc?aga?gac 576
aat?gcc?agg?aac?acc?ctg?tac?ctg?caa?atg?agc?agt?ctg?agg?tct?gag 624
gac?acg?gcc?ttg?tat?tac?tgt?gca?aga?cga?ggt?ggt?aac?tac?gcc?tgg 672
ttt?gct?tac?tgg?ggc?caa?ggg?acc?acg?gtc?acc?gtc?tcc?tca 714
The aminoacid sequence of above-mentioned anti-people aFGF-ScFv is:
Met?Thr?Gln?Ser?Pro?Ala?Ile?Leu?Ser?Ala?Ser?Pro?Gly?Glu?Lys?Val
Thr?Met?Thr?Cys?Arg?Ala?Ser?Ser?Ser?Val?Ser?Tyr?Met?His?Trp?Tyr
Gln?Gln?Lys?Pro?Gly?Ser?Ser?Pro?Lys?Pro?Trp?Ile?Tyr?Ala?Thr?Ser
Asn?Leu?Ala?Ser?Gly?Val?Pro?Ala?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly
Thr?Ser?Tyr?Ser?Leu?Thr?Ile?Ser?Arg?Val?Glu?Ala?Glu?Asp?Ala?Ala
Thr?Tyr?Tyr?Cys?Gln?Gln?Trp?Ser?Ser?Asn?Pro?Pro?Thr?Phe?Gly?Ala
Gly?Thr?Lys?Leu?Glu?Ile?Lys?Arg?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly
Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gln?Val?Lys?Leu?Gln?Gln?Ser?Gly?Gly
Gly?Leu?Val?Lys?Pro?Gly?Gly?Ser?Leu?Lys?Leu?Ser?Cys?Ala?Ala?Ser
Gly?Phe?Ala?Phe?Ser?Ser?Tyr?Asp?Met?Ser?Trp?Val?Arg?Gln?Thr?Pro
Glu?Lys?Arg?Leu?Glu?Trp?Val?Ala?Thr?Ile?Ser?Ser?Gly?Gly?Ser?Tyr
Thr?Tyr?Tyr?Pro?Asp?Ser?Val?Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp
Asn?Ala?Arg?Asn?Thr?Leu?Tyr?Leu?Gln?Met?Ser?Ser?Leu?Arg?Ser?Glu
Asp?Thr?Ala?Leu?Tyr?Tyr?Cys?Ala?Arg?Arg?Gly?Gly?Asn?Tyr?Ala?Trp
Phe?Ala?Tyr?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?*
(*: terminator codon)
The preparation of anti-people aFGF-ScFv gene may further comprise the steps:
The first step: the monoclonal antibody for preparing anti-people aFGF.
The Balb/c mouse in 8 ages in week of personnel selection aFGF immunity is merged the hybridoma cell strain that obtains the anti-people aFGF of stably excreting monoclonal antibody through splenocyte and myeloma cell.
Second step: clonal antibody variable region gene.
Extract mRNA from hybridoma cell strain, the primer that provides with Promage company antibody universal primer test kit clones by the RT-PCR method that this monoclonal antibody is light, heavy chain variable region gene.
The 3rd step: the gene fragment that obtains is cloned into business-like T carrier respectively, carries out sequential analysis,, confirm that this gene fragment is an antibody variable gene through the homology comparative analysis.
The 4th step: connect light chain of antibody, heavy chain gene, with 15 amino acid whose sons that flexibly connect, connection antibody is light, heavy chain variable region gene makes up the ScFv gene
The 5th the step: with the ScFv gene clone to business-like pET28a carrier, in the BL21 host e. coli.The conversion bacterial strain that obtains is engineering strain BL21-ScFv.
The 6th step: preparation recombinant antibodies.The culturing engineering bacterial strain, through IPTG abduction delivering 4 hours, expression efficiency reached 33%.The molecular weight that steps such as process is centrifugal, precipitation can obtain this invention is the recombinant single chain antibody of 3.0KD.Accompanying drawing is seen attached sheet.
Single-chain antibody has two antigen brachium conjunctivums, can be simultaneously and T cell and target cell combination, and activating cytotoxic T cell killing sick cell.The single-chain antibody molecular weight only is equivalent to 1/6 of complete antibody, more superior in the clinical application than complete antibody: strong a little less than the immunogenicity to the solid tumor penetrating power, can use the genetic engineering means fermentative production.Compare with traditional monoclonal antibody drug, it can carry out extensive fermentation using bacteria production, and cheap, is easy to popularization and application, is a kind of antibody formation that has application potential.
Description of drawings
Accompanying drawing is anti-people aFGF-ScFv genetic engineering bacterium expression analysis figure.
Wherein: 1, the lower molecular weight standard; 2,3, do not induce e. coli total protein; 4, IPTG induces 1 hour expressing protein; 5, IPTG induces 2 hours expressing proteins; 6, IPTG induces 3 hours expressing proteins; 7, IPTG induces 4 hours expressing proteins.Through the albumen scanning analysis, IPTG induces the expression amount of 1-4 hour scFv to account for 25%, 29%, 30% and 32% of bacterial protein respectively, satisfies the proteic requirement of engineering bacterium expression fully.
Embodiment
Embodiments of the invention comprise following content:
1. the preparation of anti-people aFGF hybridoma cell strain
Recombinant human aFGF albumen 30ug immunity female Balb/c mouse in 8 age in week, respectively at the 14th day, 35 days with the equivalent antigen immune, detected mouse tail blood with indirect elisa method on the 45th day, the mouse good to immune response carried out booster immunization at the 56th day, get its splenocyte after 3 days and merge first immunisation use Freund's complete adjuvant, all the other use Freund.
Immune mouse spleen cell is mixed with 5: 1 with murine myeloma cell NS-1, and the centrifugal 5min of 1500rpm abandons supernatant, puts in 37 ℃ of water-baths. and the PEG of 1ml preheating is slowly added, and limit edged jog is subsequently with the incomplete substratum dilution of 50ml preheating RPMI1640.The centrifugal 5min of 800rpm abandons supernatant, and the HAT substratum that adds capacity suspends again, divide kind in prepare feeder cell 96 well culture plates, the 100ul/ hole. insert 37 ℃ subsequently, 5%CO
2Cultivate in the incubator. merged the back the 7th day, changed liquid once with fresh HAT substratum half amount on the 10th day,, used the HT substratum instead, and used perfect medium instead after the 15th day in the 12nd day. and treat that cell grows at the bottom of the hole at about 1/3 o'clock, with indirect elisa method culture supernatant is detected screening. positive porocyte is carried out limited dilution cloning, antibody positive aluminium is 100% o'clock in all clone hole supernatant liquors, can carry out enlarged culturing, preserves.
2. antibody variable gene V
LAnd V
HThe clone
Take out frozen 1C9 hybridoma cell strain from liquid nitrogen container, the recovery back is cultured to logarithmic phase with the RPMI1640 that contains 20% foetal calf serum, 1000rpm, and centrifugal 5min collects 5 * 10
6Individual cell separates according to the product RNA of Amersham Biosciences company and the total RNA and the separation and purification mRNA of purification kit extraction hybridoma then.Use same company reverse transcription test kit to synthesize cDNA.
The used primer of amplification antibody variable gene derives from the ScFv phage antibody test kit of Amersham Biosciences company.In two PCR tubules, get above-mentioned synthetic cDNA 33ul respectively, in first pipe, add V
LPrimer mixture 2ul, ddH
2O 64ul. adds V in second pipe
HPrimer 1, V
HEach 2ul of primer 2, ddH
2O 62ul behind 95 ℃ of pre-sex change 5min, adds 1ul Taq archaeal dna polymerase (3u/ul).The PCR reaction conditions is: 94 ℃ of 1min, and 55 ℃ of 2min, 72 ℃ of 2min, totally 30 circulations, the PCR product reclaims the purpose fragment after 2% agarose gel electrophoresis analysis, and this purpose fragment is connected with business-like T carrier carries out sequencing.
3. the connection peptides of antibody gene VL, VH and ScFv are gene constructed
According to the aminoterminal of sequencing acquisition and the V of carboxyl terminal
LAnd V
HFurther design construction connection peptides of gene order and ScFv primer:
V
LThe positive-sense strand primer is:
5 '-CCG GAA TTC GAC ATC CAG ATG ACC CAG (
GAA TTCBe the EcoRI restriction enzyme site).
V
LThe antisense strand primer is:
5’-AGA?GCC?ACC?TCC?GCC?TGA?ACC?GCC?TCC?ACC?CCG?TTT?GAT?TTC?CAG?CTTGGT
V
HThe positive-sense strand primer is: 5 '-GGC GGA GGT GGC TCT GGC GGA GGT GGC TCT CAG GTGAAG CTG CAG C
V
HThe antisense strand primer is: 5 '-CCC
AAG CTTCTA TGA GGA GAC GGT GAC (
AAG CTTBe the HindIII restriction enzyme site).
V wherein
LAntisense strand primer and V
HItalicized item is a connection peptides in the positive-sense strand primer, and through PCR reaction amplification, the italic black matrix partly is a complementary region, designs for making up ScFv.
The PCR reaction conditions is: 94 ℃ of 5min, 94 ℃ of 1min then, 60 ℃ of 1min, 72 ℃ of 1min, 35 circulations, last 72 ℃ of 5min.After the PCR product electrophoresis recovery after extending, overlap PCR further makes up the ScFv gene.In the PCR pipe, add above-mentioned PCR product, Taq enzyme and damping fluid, under the situation that does not add primer, carry out 7 circulations: 94 ℃ of 70s, 55 ℃ of 1min, 72 ℃ of 1min. in this process because V
LAntisense strand primer and V
HComplementary region in the positive-sense strand primer, V
LAnd V
HJust can match mutually, and extend, thereby form complete V
L-connection peptides-V
HAdd primer V at last
LPositive-sense strand primer and V
HThe antisense strand primer carries out following reaction: 94 ℃ of 5min, and 94 ℃ of 70s, 60 ℃ of 1min, 72 ℃ of 1min, 72 ℃ of 2min then, 25 circulations finally obtain the ScFv gene.
4. make up the recombinant expression vector of ScFv gene
EcoRI and HindIII restriction enzyme are handled business-like pET28a (+) expression vector, by 1: 3 mixed carrier DNA of mole concentration and ScFv gene, T4DNA ligase enzyme connection carrier and ScFv gene obtain the anti-FGF-ScFv of recombinant expression vector pET28a-.
5. express the ScFv recombinant protein
The anti-FGF-ScFv of recombinant expression vector pET28a-is transformed DH5 competence intestinal bacteria, the screening of amicillin resistance substratum, the double digestion evaluation is also carried out sequential analysis.Simultaneously recombinant expression vector is transformed BL21 competence intestinal bacteria, 37 ℃ of cultivations, OD
600About 0.5, add IPTG its final concentration 0.8mM was induced 4 hours, centrifugal 10 minutes of 4 ℃ of 6000rpm, 15%SDS-PAGE analyzes recombinant antibodies.
SEQUENCE?LISTING
＜110〉Northeast Normal University
＜120〉single-chain antibody gene of human acid fibroblast growth factor resistant
<130>none
<140>none
<141>2005-11-07
<160>2
<170>PatentIn?version?3.1
<210>1
<211>714
<212>DNA
<213>Artificial?Sequence
<220>
＜223〉single-chain antibody gene of human acid fibroblast growth factor resistant
<220>
<221>CDS
<222>(1)..(714)
<223>
<220>
<221>misc_recomb
<222>(1)..(312)
＜223〉light chain gene of human acid fibroblast growth factor resistant antibody
<220>
<221>misc_recomb
<222>(313)..(357)
＜223〉connexon of connection human acid fibroblast growth factor resistant light chain of antibody and heavy chain gene
<220>
<221>misc_recomb
<222>(358)..(714)
＜223〉heavy chain gene of human acid fibroblast growth factor resistant antibody
<300>
＜302〉single-chain antibody gene of human acid fibroblast growth factor resistant
<308>none
<309>2007-05-07
<310>none
<311>2005-11-07
<312>2007-05-07
<313>(1)..(714)
<400>1
atg?acc?cag?tct?cca?gca?atc?ctg?tct?gca?tct?cca?ggg?gag?aag?gtc 48
Met?Thr?Gln?Ser?Pro?Ala?Ile?Leu?Ser?Ala?Ser?Pro?Gly?Glu?Lys?Val
1 5 10 15
aca?atg?act?tgc?agg?gcc?agc?tca?agt?gta?agt?tac?atg?cac?tgg?tac 96
Thr?Met?Thr?Cys?Arg?Ala?Ser?Ser?Ser?Val?Ser?Tyr?Met?His?Trp?Tyr
20 25 30
cag?cag?aag?cca?gga?tcc?tcc?ccc?aaa?ccc?tgg?att?tat?gcc?aca?tcc 144
Gln?Gln?Lys?Pro?Gly?Ser?Ser?Pro?Lys?Pro?Trp?Ile?Tyr?Ala?Thr?Ser
35 40 45
aac?ctg?gct?tct?gga?gtc?cct?gct?cgc?ttc?agt?ggc?agt?ggg?tct?ggg 192
Asn?Leu?Ala?Ser?Gly?Val?Pro?Ala?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly
50 55 60
acc?tct?tac?tct?ctc?aca?atc?agc?aga?gtg?gag?gct?gaa?gat?gct?gcc 240
Thr?Ser?Tyr?Ser?Leu?Thr?Ile?Ser?Arg?Val?Glu?Ala?Glu?Asp?Ala?Ala
65 70 75 80
act?tat?tac?tgc?cag?cag?tgg?agt?agt?aac?cca?ccc?acg?ttc?ggt?gct 288
Thr?Tyr?Tyr?Cys?Gln?Gln?Trp?Ser?Ser?Asn?Pro?Pro?Thr?Phe?Gly?Ala
85 90 95
ggc?acc?aag?ctg?gaa?atc?aaa?cgg?ggt?gga?ggc?ggt?tca?ggc?gga?ggt 336
Gly?Thr?Lys?Leu?Glu?Ile?Lys?Arg?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly
100 105 110
ggc?tct?ggc?gga?ggt?ggc?tct?cag?gtg?aag?ctg?cag?cag?tca?ggg?gga 384
Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gln?Val?Lys?Leu?Gln?Gln?Ser?Gly?Gly
115 120 125
ggc?tta?gtg?aag?cct?gga?ggg?tcc?ctg?aaa?ctc?tcc?tgt?gca?gcc?tct 432
Gly?Leu?Val?Lys?Pro?Gly?Gly?Ser?Leu?Lys?Leu?Ser?Cys?Ala?Ala?Ser
130 135 140
gga?ttc?gct?ttc?agt?agc?tat?gac?atg?tct?tgg?gtt?cgc?cag?act?ccg 480
Gly?Phe?Ala?Phe?Ser?Ser?Tyr?Asp?Met?Ser?Trp?Val?Arg?Gln?Thr?Pro
145 150 155 160
gag?aag?agg?ctg?gag?tgg?gtc?gca?acc?att?agt?agt?ggt?ggt?agt?tac 528
Glu?Lys?Arg?Leu?Glu?Trp?Val?Ala?Thr?Ile?Ser?Ser?G]y?Gly?Ser?Tyr
165 170 175
acc?tac?tat?cca?gac?agt?gtg?aag?ggc?cga?ttc?acc?atc?tcc?aga?gac 576
Thr?Tyr?Tyr?Pro?Asp?Ser?Val?Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp
180 185 190
aat?gcc?agg?aac?acc?ctg?tac?ctg?caa?atg?agc?agt?ctg?agg?tct?gag 624
Asn?Ala?Arg?Asn?Thr?Leu?Tyr?Leu?Gln?Met?Ser?Ser?Leu?Arg?Ser?Glu
195 200 205
gac?acg?gcc?ttg?tat?tac?tgt?gca?aga?cga?ggt?ggt?aac?tac?gcc?tgg 672
Asp?Thr?Ala?Leu?Tyr?Tyr?Cys?Ala?Arg?Arg?Gly?Gly?Asn?Tyr?Ala?Trp
210 215 220
ttt?gct?tac?tgg?ggc?caa?ggg?acc?acg?gtc?acc?gtc?tcc?tca 714
Phe?Ala?Tyr?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?*
225 230 235
<210>2
<211>238
<212>PRT
<213>Artificial?Sequence
<220>
＜223〉single-chain antibody gene of human acid fibroblast growth factor resistant
<400>2
Met?Thr?Gln?Ser?Pro?Ala?Ile?Leu?Ser?Ala?Ser?Pro?Gly?Glu?Lys?Val
1 5 10 15
Thr?Met?Thr?Cys?Arg?Ala?Ser?Ser?Ser?Val?Ser?Tyr?Met?His?Trp?Tyr
20 25 30
Gln?Gln?Lys?Pro?Gly?Ser?Ser?Pro?Lys?Pro?Trp?Ile?Tyr?Ala?Thr?Ser
35 40 45
Asn?Leu?Ala?Ser?Gly?Val?Pro?Ala?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly
50 55 60
Thr?Ser?Tyr?Ser?Leu?Thr?Ile?Ser?Arg?Val?Glu?Ala?Glu?Asp?Ala?Ala
65 70 75 80
Thr?Tyr?Tyr?Cys?Gln?Gln?Trp?Ser?Ser?Asn?Pro?Pro?Thr?Phe?Gly?Ala
85 90 95
Gly?Thr?Lys?Leu?Glu?Ile?Lys?Arg?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly
100 105 110
Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gln?Val?Lys?Leu?Gln?Gln?Ser?Gly?Gly
115 120 125
Gly?Leu?Val?Lys?Pro?Gly?Gly?Ser?Leu?Lys?Leu?Ser?Cys?Ala?Ala?Ser
130 135 140
Gly?Phe?Ala?Phe?Ser?Ser?Tyr?Asp?Met?Ser?Trp?Val?Arg?Gln?Thr?Pro
145 150 155 160
Glu?Lys?Arg?Leu?Glu?Trp?Val?Ala?Thr?Ile?Ser?Ser?Gly?Gly?Ser?Tyr
165 170 175
Thr?Tyr?Tyr?Pro?Asp?Ser?Val?Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp
180 185 190
Asn?Ala?Arg?Asn?Thr?Leu?Tyr?Leu?Gln?Met?Ser?Ser?Leu?Arg?Ser?Glu
195 200 205
Asp?Thr?Ala?Leu?Tyr?Tyr?Cys?Ala?Arg?Arg?Gly?Gly?Asn?Tyr?Ala?Trp
210 215 220
Phe?Ala?Tyr?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?Val?Ser *
225 230 235
Claims (1)
1, the single-chain antibody gene of human acid fibroblast growth factor resistant is characterized in that the VL and the VH gene that are connected antibody by connection peptides set up, and this gene is by 714 based compositions:
The nucleotides sequence of this gene is classified as:
atg?acc?cag?tct?cca?gca?atc?ctg?tct?gca?tct?cca?ggg?gag?aag?gtc 48
aca?atg?act?tgc?agg?gcc?agc?tca?agt?gta?agt?tac?atg?cac?tgg?tac 96
cag?cag?aag?cca?gga?tcc?tcc?ccc?aaa?ccc?tgg?att?tat?gcc?aca?tcc 144
aac?ctg?gct?tct?gga?gtc?cct?gct?cgc?ttc?agt?ggc?agt?ggg?tct?ggg 192
acc?tct?tac?tct?ctc?aca?atc?agc?aga?gtg?gag?gct?gaa?gat?gct?gcc 240
act?tat?tac?tgc?cag?cag?tgg?agt?agt?aac?cca?ccc?acg?ttc?ggt?gct 288
ggc?acc?aag?ctg?gaa?atc?aaa?cgg?ggt?gga?ggc?ggt?tca?ggc?gga?ggt 336
ggc?tct?ggc?gga?ggt?ggc?tct?cag?gtg?aag?ctg?cag?cag?tca?ggg?gga 384
ggc?tta?gtg?aag?cct?gga?ggg?tcc?ctg?aaa?ctc?tcc?tgt?gca?gcc?tct 432
gga?ttc?gct?ttc?agt?agc?tat?gac?atg?tct?tgg?gtt?cgc?cag?act?ccg 480
gag?aag?agg?ctg?gag?tgg?gtc?gca?acc?att?agt?agt?ggt?ggt?agt?tac 528
acc?tac?tat?cca?gac?agt?gtg?aag?ggc?cga?ttc?acc?atc?tcc?aga?gac 576
aat?gcc?agg?aac?acc?ctg?tac?ctg?caa?atg?agc?agt?ctg?agg?tct?gag 624
gac?acg?gcc?ttg?tat?tac?tgt?gca?aga?cga?ggt?ggt?aac?tac?gcc?tgg 672
ttt?gct?tac?tgg?ggc?caa?ggg?acc?acg?gtc?acc?gtc?tcc?tca 714
The aminoacid sequence of this genes encoding is:
Met?Thr?Gln?Ser?Pro?Ala?Ile?Leu?Ser?Ala?Ser?Pro?Gly?Glu?Lys?Val
Thr?Met?Thr?Cys?Arg?Ala?Ser?Ser?Ser?Val?Ser?Tyr?Met?His?Trp?Tyr
Gln?Gln?Lys?Pro?Gly?Ser?Ser?Pro?Lys?Pro?Trp?Ile?Tyr?Ala?Thr?Ser
Asn?Leu?Ala?Ser?Gly?Val?Pro?Ala?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly
Thr?Ser?Tyr?Ser?Leu?Thr?Ile?Ser?Arg?Val?Glu?Ala?Glu?Asp?Ala?Ala
Thr?Tyr?Tyr?Cys?Gln?Gln?Trp?Ser?Ser?Asn?Pro?Pro?Thr?Phe?Gly?Ala
Gly?Thr?Lys?Leu?Glu?Ile?Lys?Arg?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly
Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gln?Val?Lys?Leu?Gln?Gln?Ser?Gly?Gly
Gly?Leu?Val?Lys?Pro?Gly?Gly?Ser?Leu?Lys?Leu?Ser?Cys?Ala?Ala?Ser
Gly?Phe?Ala?Phe?Ser?Ser?Tyr?Asp?Met?Ser?Trp?Val?Arg?Gln?Thr?Pro
Glu?Lys?Arg?Leu?Glu?Trp?Val?Ala?Thr?Ile?Ser?Ser?Gly?Gly?Ser?Tyr
Thr?Tyr?Tyr?Pro?Asp?Ser?Val?Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp
Asn?Ala?Arg?Asn?Thr?Leu?Tyr?Leu?Gln?Met?Ser?Ser?Leu?Arg?Ser?Glu
Asp?Thr?Ala?Leu?Tyr?Tyr?Cys?Ala?Arg?Arg?Gly?Gly?Asn?Tyr?Ala?Trp
Phe?Ala?Tyr?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?Val?Ser *。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005101190451A CN100345966C (en) | 2005-11-30 | 2005-11-30 | Human acid fibroblast growth factor resistant single-chain antibody gene |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005101190451A CN100345966C (en) | 2005-11-30 | 2005-11-30 | Human acid fibroblast growth factor resistant single-chain antibody gene |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1800394A CN1800394A (en) | 2006-07-12 |
| CN100345966C true CN100345966C (en) | 2007-10-31 |
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| CNB2005101190451A Expired - Fee Related CN100345966C (en) | 2005-11-30 | 2005-11-30 | Human acid fibroblast growth factor resistant single-chain antibody gene |
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| CN105214088A (en) * | 2015-11-06 | 2016-01-06 | 东北师范大学 | The medical application of humanization aFGF single-chain antibody in treatment tumor |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH02306996A (en) * | 1988-07-04 | 1990-12-20 | Takeda Chem Ind Ltd | Antibody of acidic fibroblast cell growth factor, its use and peptide thereof |
| JPH03228691A (en) * | 1990-02-05 | 1991-10-09 | Rikagaku Kenkyusho | monoclonal antibody |
| US5437995A (en) * | 1989-09-26 | 1995-08-01 | Takeda Chemical Industries, Ltd. | Monoclonal antibody against an acidic FGF protein and hybridoma for its production |
-
2005
- 2005-11-30 CN CNB2005101190451A patent/CN100345966C/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH02306996A (en) * | 1988-07-04 | 1990-12-20 | Takeda Chem Ind Ltd | Antibody of acidic fibroblast cell growth factor, its use and peptide thereof |
| US5437995A (en) * | 1989-09-26 | 1995-08-01 | Takeda Chemical Industries, Ltd. | Monoclonal antibody against an acidic FGF protein and hybridoma for its production |
| JPH03228691A (en) * | 1990-02-05 | 1991-10-09 | Rikagaku Kenkyusho | monoclonal antibody |
Non-Patent Citations (1)
| Title |
|---|
| 人酸性成纤维细胞生长因子单克隆抗体的制备 王芳，薛沿宁，王会信，徐秀英，刘农乐，邵宁生，周延冲，免疫学杂志，第9卷第4期 1993 * |
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| CN1800394A (en) | 2006-07-12 |
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