CN101717775B - Single-chain antibody gene of anti-human death receptor 5 - Google Patents
Single-chain antibody gene of anti-human death receptor 5 Download PDFInfo
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Abstract
The invention discloses a single-chain antibody gene of anti-human death receptor 5 and relates to a gene. The invention also provides a single-chain antibody (aDR5-scFv) gene of anti-human death receptor 5 and a preparation method and applications thereof. The single-chain antibody of the anti-human death receptor 5 is established by linking light chain (VL) gene with heavy chain (VH) gene of the antibody by a linker and is composed of 726 bases. The preparation method comprises: a step of preparing monoclonal antibody of anti-human DR5; a step of cloning variable region gene of the antibody; a step of implementing sequencing analysis on resultant genetic fragments; a step of using the linker to link the light chain variable region gene with the heavy chain variable region gene; a step of constructing expression vector; and a step of preparing recombinant antibody. The invention further provides the application of the single-chain antibody of the anti-human death receptor 5 in preparing antitumor medicines.
Description
Technical field
The present invention relates to a kind of gene, especially relate to a kind of anti-human death receptor 5 (death receptor 5, single-chain antibody gene DR5) and recombinant vectors thereof and expression product.
Background technology
The appearance of single-chain antibody is the intensification of people's antagonist understanding and the inevitable outcome of scientific technological advance.Since Britain and Japanese scholar have found Diphtheria Antitoxin, and since first immune serum being used for the passive immunotherapy and serodiagnosis of transmissible disease, antibody technique enjoys people to pay close attention to as a kind of treatment means.
1975, (Kohler and Milstein.Nature, 1975 such as Kohler; 256:495) find and utilize hybridoma technology; Successfully prepare monoclonal antibody, overcome the shortcoming of polyclonal antibody poor specificity, so the appearance of monoclonal antibody gets more and more people's extensive concerning with high specific and avidity; The various countries scholar puts in the studies on Monoclonal Antibody work one after another, and the effective ways of diagnosis and treatment disease are found in expectation.At present, there has been multiple monoclonal antibody to get into clinical use.But the monoclonal antibody of clinical application at present basically all is the mouse source, and the progress of human monoclonal antibody is slow.
Though monoclonal antibody technique has obtained very big development; But still exist limitation; Wherein the most fatal defective is that monoclonal antibody causes immunological rejection as a kind of heterologous protein entering people cognition; This has not only influenced the performance of monoclonal antibody curative effect, also can disturb its proper distribution in vivo, thereby limits its application in clinical.
After getting into the eighties in 20th century, the fast development of Protocols in Molecular Biology has promoted the progress of antibody technique research, and genetic engineering antibody arises at the historic moment, and wherein paid close attention to by people with single-chain antibody.Since (Bird andHuston such as Bird in 1988; Procatl Acad Sci USA; 1988,5:5875-5879) successfully developed since first single-chain antibody, succeeded in developing and produced multiple single-chain antibody at present; Except that the in-vitro diagnosis that is used for basic medical research and disease, there are some to begin to be used for clinical treatment.As an emerging things, the single-chain antibody technology has become the forward position and the hot issue of antibody Related Research Domain now.It not only can be transformed existing antibody, can also create the non-existent brand-new antibody of nature, both can reduce the immunogenicity of antibody, can also strengthen the curative effect of antibody.
Single-chain antibody is the small molecular antibody with the gene engineering method preparation, is by the variable region of heavy chain (V of elasticity connection peptides (being generally 12～15 amino acid) with antibody
H) and variable region of light chain (V
L) recombinant antibodies that is formed by connecting; Its molecular weight only is equivalent to the sixth of original antibody; But single-chain antibody contains whole antigen binding sites, so single-chain antibody has farthest kept the antigen-binding activity of antibody, is the minimal segment with parental antibody antigen-binding activity.
After single-chain antibody occurs; People are in the process of research single-chain antibody; Find some unrivaled meliority that single-chain antibody has; So people just utilize prior art that single-chain antibody is transformed and modified; With the function purposes new with development of improving single-chain antibody, the new antibodies that develops based on single-chain antibody that has occurred at present has following several kinds: single-chain antibody polymer, bispecific single-chain antibody, single domain antibody, the single-chain antibody with constant region, intracellular antibody (intracellular antibodyor intrabody).Wherein the intracellular antibody technology is in recent years, the further investigation of signal conduction in Along with people's antagonist engineering and the cell, the antibody technique of important target protein in the derivative brand-new cell capable of blocking.Thereby the development of single-chain antibody technology and deriving technology thereof is for diagnosis, treatment and the research of tumour, immune correlated disease are laid a good foundation.
TRAIL TRAIL (Tumor necrosis factor-Related Apoptosis InducingLigand) is the another member of the TNF superfamily found recently.Different with other members of TNF family is that TRAIL only exists with film mating type in vivo.Experiment in vitro shows, no matter film mating type, the still TRAIL of solvable type, the cell generation apoptosis of abduction delivering TRAIL specific receptors rapidly.Experiment in vivo and vitro shows that TRAIL combines with death receptor optionally to induce the kinds of tumor cells apoptosis, and most of normal cells are not had tangible lethal effect.This characteristic makes its other member who is superior to tnf ligand family, becomes the new power of oncotherapy.
DR5 claims TRAIL-R2 again, belongs to I type transmembrane protein, is rich in the structural domain and the death domain of halfcystine.Multinomial achievement in research shows; DR5 has expression in normal cell and tumour cell; Like liver, lung fetus; And adult's the medium position of lymphocyte, liver, ovary, spleen and lung, be expressed in many tumor tissues, like thyroid carcinoma, laryngocarcinoma, lung cancer, liver cancer, mammary cancer, carcinoma of the pancreas, carcinoma of testis, prostate cancer, ovarian cancer, cervical cancer, uterus carcinoma, the rectum cancer etc. especially high-levelly.
Nineteen ninety-five, Wiley was at Immunity; 1995; 3 (6): clone and found be correlated with tumour necrosis factor (Tumor necrosis factor related apoptosis-inducing ligand, TRAIL) gene confirm also called after Apo-2L again by Pitti etc. to inducing ligand in 673.The TRAIL wide expression like peripheral blood lymphocyte, lung, kidney, spleen, thymus gland, prostate gland, ovary, small intestine, heart, placenta, Skelettmuskel etc., and does not detect expression in normal people's various tissues in brain, liver and testis.Present known TRAIL has two kinds of forms: film mating type TRAIL (TRAIL of total length) and solvable type TRAIL (168 amino acid of extracellular region C end), and they are at the external equal apoptosis that can induce kinds of tumor cells, and reach is very wide.
Calendar year 2001, Ichikawa was at Nat.Med.; 2001; Prepare among the 7:954 the special competitive monoclonal antibody of people DR5 (Agonistic monoclonal antibody specific for human DR5, TRA-8), this is death receptor DR5 and synthetic monoclonal antibody that specificity is directed against people's surface of cell membrane; It is different with the monoclonal antibody (Monoclonalantibody against human DR5) of general anti-people DR5; It can combine with death receptor DR5 specifically, the effect of performance part, thereby cell death inducing.The process for preparing this monoclonal antibody is the cell outskirt of people DR5 and human IgG1's (DR5-Ig) Fc section to be formed the immune female BALB/c mouse of fusion rotein, thereby synthesized this monoclonal antibody of TRA-8.Experiment showed, that TRA-8 can induce the apoptosis of the tumour cell of many expression DR5 acceptors, and to the normal tissue cell nontoxicity, and with the analysis of Western-blot method, TRA-8 not can with other death receptor generation cross reaction.
Because death receptor DR5 is in the uniqueness of cell distribution and mechanism of action; On the direction of research apoptosis of tumor cells, good prospect is provided for people; Along with further investigation, it is found that on anticancer therapy, also to exist some problem, like the resistance of TRAIL to liver cancer to death receptor DR5 and part thereof; And it is to hepatocellular toxicity etc., has become the problem that people put forth effort to inquire at present.And TRA-8 also is in the middle of the development; But continuous exploration to the new part of DR5 acceptor; Deepened people to the DR5 acceptor in the understanding aspect generation, development and the treatment of apoptosis of tumor cells, and wide theoretical foundation is provided for brand-new oncotherapy scheme.
Summary of the invention
Single-chain antibody (aDR5-scFv) gene that the purpose of this invention is to provide anti-human death receptor 5.
Another object of the present invention provides the preparation method of the single-chain antibody gene of said anti-human death receptor 5.
Another object of the present invention provides the single-chain antibody gene of described anti-human death receptor 5 and in the preparation antitumor drug, uses.
The single-chain antibody gene of said anti-human death receptor 5 is the light chain (V that is connected antibody by connection peptides
L) and heavy chain (V
H) gene sets up, the single-chain antibody gene of said anti-human death receptor 5 is by 726 based compositions,
Its nucleotides sequence is classified as:
1 atg?gga?cag?gtc?cag?ctg?cag?caa?tct?gga?cct?gag?ctg?gtg?aag?cct
49 ggg?gct?tca?gtg?aag?ata?tcc?tgc?aag?gct?tct?ggc?tat?acc?ttc?aca
97 agc?tac?tat?ata?cac?tgg?gtg?aag?cag?agg?cct?gga?cag?ggc?ctt?gag
145 tgg?att?gga?tat?att?tat?cct?aga?gat?ggt?agt?act?aat?tac?aat?gag
193 aag?ttc?aag?ggc?aag?gcc?aca?ctg?act?gca?gac?aca?tcc?tcc?agc?aca
241 gcc?tac?atg?cag?ctc?agc?agc?ctg?aca?tct?gag?gac?tct?gca?gtc?tat
289 ttc?tgt?gca?agg?gcc?ctc?tat?gat?ggt?cac?tac?gtg?ggg?gct?atg?gac
337 tac?tgg?ggt?caa?gga?gga?ggt?ggc?gga?tct?gga?ggg?ggt?ggt?agc?ggt
385 gga?ggc?ggg?agt?ggg?gcc?att?tcc?cag?gct?gtt?gtg?act?cag?gaa?tct
433 gca?ctc?acc?aca?tca?cct?ggt?gaa?aca?gtc?aca?ctc?act?tgt?cgc?tca
481 agt?act?ggg?gct?gtt?aca?acc?agt?aac?tat?gcc?aac?tgg?gtc?caa?gaa
529 aaa?cca?gat?cat?tta?ttc?act?ggt?cta?ata?ggt?ggt?acc?aac?aac?cga
577 gct?cca?ggt?gtt?cct?gcc?aga?atc?tca?ggc?tcc?ctg?att?gga?gac?aag
625 gct?gcc?ctc?acc?atc?aca?ggg?gct?cag?act?gag?gat?gag?gca?ata?tat
673 ttc?tgt?gtt?cta?tgg?tac?agc?aac?cat?tgg?gtg?ttc?ggt?gga?gga?acc
721 aaa?ctg
Its aminoacid sequence is:
1 MET?Gly?Gln?Val?Gln?Leu?Gln?Gln?Ser?Gly?Pro?Glu?Leu?Val?Lys?Pro
17 Gly?Ala?Ser?Val?Lys?Ile?Ser?Cys?Lys?Ala?Ser?Gly?Tyr?Thr?Phe?Thr
33 Ser?Tyr?Tyr?Ile?His?Trp?Val?Lys?Gln?Arg?Pro?Gly?Gln?Gly?Leu?Glu
49 Trp?Ile?Gly?Tyr?Ile?Tyr?Pro?Arg?Asp?Gly?Ser?Thr?Asn?Tyr?Asn?Glu
65 Lys?Phe?Lys?Gly?Lys?Ala?Thr?Leu?Thr?Ala?Asp?Thr?Ser?Ser?Ser?Thr
81 Ala?Tyr?MET?Gln?Leu?Ser?Ser?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Val?Tyr
97 Phe?Cys?Ala?Arg?Ala?Leu?Tyr?Asp?Gly?His?Tyr?Val?Gly?Ala?MET?Asp
113 Tyr?Trp?Gly?Gln?Gly?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly
129 Gly?Gly?Gly?Ser?Gly?Ala?Ile?Ser?Gln?Ala?Val?Val?Thr?Gln?Glu?Ser
145 Ala?Leu?Thr?Thr?Ser?Pro?Gly?Glu?Thr?Val?Thr?Leu?Thr?Cys?Arg?Ser
161 Ser?Thr?Gly?Ala?Val?Thr?Thr?Ser?Asn?Tyr?Ala?Asn?Trp?Val?Gln?Glu
177 Lys?Pro?Asp?His?Leu?Phe?Thr?Gly?Leu?Ile?Gly?Gly?Thr?Asn?Asn?Arg
193 Ala?Pro?Gly?Val?Pro?Ala?Arg?Ile?Ser?Gly?Ser?Leu?Ile?Gly?Asp?Lys
209 Ala?Ala?Leu?Thr?Ile?Thr?Gly?Ala?Gln?Thr?Glu?Asp?Glu?Ala?Ile?Tyr
225 Phe?Cys?Val?Leu?Trp?Tyr?Ser?Asn?His?Trp?Val?Phe?Gly?Gly?Gly?Thr
241Lys?Leu?＊
(*: terminator codon)
The preparation method of the single-chain antibody gene of said anti-human death receptor 5 may further comprise the steps:
1) step of the monoclonal antibody of an anti-people DR5 of preparation;
2) step of a clonal antibody variable region gene;
3) step of the gene fragment that obtains being carried out sequencing analysis;
4) step that connects light chain of antibody, heavy chain variable region gene with connection peptides (Linker);
5) step of a construction of expression vector;
6) step of a preparation recombinant antibodies.
The step of the monoclonal antibody of the anti-people DR5 of said preparation is the personnel selection DR5 protein immunization Balb/c mouse in 8 ages in week, merges the hybridoma cell strain that obtains stably excreting anti-people DR5 monoclonal antibody (aDR5 mAb) through splenocyte and myeloma cell.
The step of said clonal antibody variable region gene is from hybridoma cell strain, to extract mRNA, and the primer that provides with Novagen company antibody universal primer test kit clones through the RT-PCR method that this monoclonal antibody is light, heavy chain variable region gene.
The step that said gene fragment to acquisition is carried out sequencing analysis is that the gene fragment that obtains is cloned into business-like T carrier (pMD19-T simple vector respectively; TaKaRa); Carry out sequential analysis,, confirm that this gene fragment is an antibody variable gene through the homology comparative analysis.
Said step with connection peptides (Linker) connection light chain of antibody, heavy chain variable region gene is the design primer, and antibody is light with 15 amino acid whose flexible peptide linkers, heavy chain variable region gene couples together, and is built into the scFv gene.
The step of said construction of expression vector is that the scFv gene clone is arrived on business-like pET-22b (+) carrier, transforms the Rosetta-gami escherichia coli host, and obtaining to transform bacterial strain is engineering strain Rosetta-gami-aDR5-scFv.
The step of said preparation recombinant antibodies is that through IPTG abduction delivering 12h, expression efficiency reaches more than 30% with LB liquid nutrient medium culturing engineering bacterial strain.After passing through step purifying such as broken bacterium, mistake ni-sepharose purification, the molecular weight that obtains this invention is the recombinant single chain antibody of 30kD.
The single-chain antibody gene of said anti-human death receptor 5 can be used in the preparation antitumor drug.
Said single-chain antibody has two antigen brachium conjunctivums, can combine with T cell and target cell simultaneously, and activating cytotoxic T cell killing sick cell.The single-chain antibody molecular weight only is equivalent to 1/6 of complete antibody, and is more superior than complete antibody in clinical application, strong to the solid tumor penetrating power a little less than the immunogenicity, available genetic engineering means fermentative prodn.Compare with traditional monoclonal antibody drug, can carry out extensive fermentation using bacteria production, price is low, is easy to popularization and application, is a kind of antibody formation that has application potential.
Anti-DR5 single-chain antibody provided by the invention (aDR5-scFv) be single-chain antibody that actual application value is arranged (single-chainFv, scFv).ADR5-scFv is with aDR5 mAb light chain (V by a connection peptides
L) and heavy chain (V
H) the gene formed single peptide chain that connects together; Through correct folding; Two variable regions form the fragment with antigen combined function by non covalent bond, and the aDR5-scFv molecular weight is little, are prone to penetrate solid tissue and inside tumor; And keep the characteristic and the function of parental antibody conjugated antigen, thereby be a kind of immunity therapeutic preparation with potential using value.
Description of drawings
Fig. 1 is anti-people DR5 single-chain antibody (aDR5-scFv) genetic engineering bacterium expression analysis figure.In Fig. 1,1, do not induce e. coli total protein; 2, IPTG induces the 1h expressing protein; 3, IPTG induces the 3h expressing protein; 4, IPTG induces the 6h expressing protein; 5, IPTG induces the 12h expressing protein; 6, the scFv behind the purifying; 7, the molecular weight of albumen standard.Through the albumen scanning analysis, IPTG induces the expression amount of 1～12h scFv to account for 24%, 28%, 33%, 35% of bacterial protein respectively, satisfies the proteic requirement of engineering bacterium expression fully.
Embodiment
1. the preparation of anti-people DR5 hybridoma cell strain
Recombinant human DR5 protein 10 0 μ L (about 45 μ g) carries out emulsification with the equivalent Freund's complete adjuvant, is fully mixed to the water-in-oil shape.In the subcutaneous multi-point injection of 8 week female Balb/c mouse backs in age.Whenever add equivalent Freund's incomplete adjuvant booster immunization 2 times at a distance from 2 weeks with the DR5 of same dose.After 15 days, abdominal injection people DR5 (50 μ g) and Freund's incomplete adjuvant mixture.Last booster immunization 4 days is after eyeball is got blood, and indirect elisa method is measured tiring of anti-sDR5 antibody in the mice serum, selects the high mouse of antiserum titre through tail vein injection recombinant human DR5 protein 10 0 μ L booster immunization, carries out cytogamy after 3 days.
Behind the booster immunization 3 days, get the high immune Balb/c mouse of antiserum titre, after eyeball is got blood (get serum and give over to positive control); Take off neck and put to death mouse, 75% alcohol-pickled sterilization skin, the aseptic spleen of getting; Squeeze out splenocyte, add 20mL GKN solution in plate, the 10mL suction pipe dispels the splenocyte agglomerate; Cell suspension is moved to the 50mL centrifuge tube, the preparation immune spleen cell.Get myeloma cell Sp2/0 in another centrifuge tube, after the centrifugal 5min of 1500r/min abandons supernatant, add GKN solution 20mL respectively, counting, the centrifugal 5min of 1500r/min simultaneously.According to count results, the adjustment splenocyte: myeloma cell=6: 1, and splenocyte concentration is 3 * 10
6/ mL.After mixing two kinds of cells, 1500r/min is centrifugal, and 5min abandons supernatant, and bullet looses and manages floor cells; Centrifuge tube places 37 ℃ of water-baths; Slowly add 50%PEG solution 1mL (the limit edged stirs gently) in the 1min, leave standstill 40s, add 1mL GKN solution (the limit edged stirs gently) in the 1min; In 2min, to add 5mLGKN again, add 10mL GKN solution in the 2min subsequently with method.With the centrifugal 7min of above-mentioned cell suspension 800r/min, abandon supernatant, bullet looses and manages floor cells gently, adds required 20%HAT nutrient solution, adds behind the mixing and has spread in 96 orifice plates of nurse cell, places 37 ℃ of CO
2Cultivate in the incubator.
Merge and added the HAT nutrient solution in back second day.Change liquid once after merging a week, add RPMI 1640 complete culture solutions that contain HAT.HAT uses the HT nutrient solution instead and cultivated for two weeks after selecting nutrient solution to keep two weeks of cultivation, next uses general nutrient solution instead.(about the 10th day) detection specificity production of antibodies during 1/10 area at the bottom of hybridoma is covered with the hole.With sDR5 albumen coated elisa plate, indirect elisa method screening positive clone.
Detecting the same day that screens positive hybridoma through ELISA promptly adopts limiting dilution assay that positive hole hybridoma is carried out cloning.In cloning previous day, prepare feeder cell with the normal mouse spleen earlier, spread 96 orifice plates, the same fusion experiment of preparation method.After blowing and beating evenly repeatedly with sample injector the hybridoma of waiting to do cloning in 96 orifice plates, get a little cell suspension to another sterile test tube, the cell of getting in this test tube carries out accurate counting.Get 300 cell suspensions (about 5 cells/0.1mL), inoculate 96 well culture plates, 0.1mL/ hole, totally 32 holes in the 6mL nutrient solution; Remaining 2.8mL adds the 3.2mL nutrient solution again, altogether 6mL (inoculation 96 well culture plates of about 2.5 cells/0.1mL); 0.1mL/ the hole, totally 32 holes remain 2.8mL at last; Add the 3.2mL nutrient solution again, altogether 6mL (about 1.25 cells/0.1mL), inoculate 32 well culture plate 0.1mL/ holes.Culture plate placed contain 5%CO
237 ℃ of incubators in cultivate, examine under a microscope cell clone about 5 days.Cultivate after 7～10 days, ELISA detects screening positive clone, cloning once more.After cloning detected twice 100% positives, the positive monoclonal hybridoma that picking is tired high changed 24 porocyte culture plates over to and carries out enlarged culturing, and frozen several the monoclonal cells of palpus are subsequent use simultaneously.
2. antibody variable gene V
LAnd V
HThe clone
But filter out the hybridoma cell strain of the monoclonal antibody of a strain secretion inducing apoptosis of tumour cell, be cultured to logarithmic phase with the RPMI 1640 that contains 10% foetal calf serum, 1000rpm, centrifugal 5min collects 5 * 10
6Individual cell extracts the total RNA and the separation and purification mRNA of hybridoma then according to " the RNA separation and purification test kit " of AmershamBiosciences company, the primer that uses Novagen company antibody universal primer test kit to provide carries out rt and PCR.
The rt condition is: behind 70 ℃ of insulation 10min rapidly at chilling 5min on ice, 42 ℃ of insulation 1h, cooled on ice behind 70 ℃ of insulation 15min, the cDNA that obtains directly is used for pcr amplification.The PCR condition is: 94 ℃ of insulation 1min, and 58 ℃ of insulation 50s, 72 ℃ of insulation 2min, totally 30 circulations, the PCR product reclaims the purpose fragment after 2% agarose gel electrophoresis analysis, and is connected with pMD-T Simple Vector, and order-checking is identified.
3. antibody gene V
H, V
LConnection peptides and scFv gene constructed
V according to the sequencing acquisition
HWith V
LThe aminoterminal of gene order and carboxyl terminal sequence, the primer of design construction scFv, and bring the connection peptides sequence into through primer:
V
HThe positive-sense strand primer is:
5′-AAAA
CCATGGGACAGGTCCAGCTGCAGCAATCTGGACCTGAG-3′
(
CCATGGBe Nco I restriction enzyme site)
V
HThe antisense strand primer is:
5′-
TCCTTGACCCCAGTAGTCCATAGCC-3′
V
LThe positive-sense strand primer is:
5′-
GGGGCCATTTCCCAGGCTGT-3′
V
LThe antisense strand primer is:
5′-AAAA
CTCGAGCAGTTTGGTTCCTCCACCGAACACCCAATGG-3′
(
CTCGAGBe Xho I restriction enzyme site)
Wherein, black matrix partly is a connection peptides in VH antisense strand primer and the VL positive-sense strand primer, and through PCR reaction amplification, the italic black matrix partly is a complementary sequence, designs for making up scFv.
The PCR reaction conditions is: 94 ℃ of 5min; 94 ℃ of 1min then, 60 ℃ of 50s, 72 ℃ of 2min, 30 circulations; Last 72 ℃ of 10min.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 10 circulations: 94 ℃ of 2min, 58 ℃ of 1min, 72 ℃ of 1min.In this process, V
HAntisense strand primer and V
LThe complementary sequence of positive-sense strand primer carries out complementary pairing, extends the back and forms complete VH-connection peptides-VL.Add VH positive-sense strand primer at last and VL antisense strand primer carries out following reaction: 94 ℃ of 5min; 94 ℃ of 1min, 60 ℃ of 50s, 72 ℃ of 1min, 30 circulations; 72 ℃ of 10min finally obtain the scFv gene order.
4. make up the recombinant expression vector of scFv gene
Restriction enzyme Nco I and Xho I digest expression vector pET-22b (+) and aDR5-scFv gene order respectively; After the purifying and recovering by 1: 10 mixed expression vector of mole concentration and scFv gene; With T4 dna ligase connection carrier and scFv gene, obtain recombinant expression vector pET-22b (+)-aDR5-scFv.
5. express the aDR5-scFv recombinant protein
Recombinant expression vector pET-22b (+)-aDR5-scFv is transformed DH5 α competence intestinal bacteria, and penbritin LB liquid nutrient medium is cultivated screening, and double digestion is identified and the evaluation of checking order.The positive colony enlarged culturing is extracted the expression vector plasmid and is transformed Rosetta-gami competence intestinal bacteria, and 37 ℃ of cultivations are when being cultured to OD
600Be about at 0.6 o'clock, it is 0.6mM that adding IPTG makes its final concentration, induces 12h.4 ℃ of centrifugal 20min of 8000rpm collect thalline, and the SDS-PAGE with 15% analyzes aDR5-scFv expression of recombinant proteins situation.
Sequence table
The nucleotides sequence of the single-chain antibody of anti-human death receptor 5 (aDR5-scFv) gene is classified as:
1 atg?gga?cag?gtc?cag?ctg?cag?caa?tct?gga?cct?gag?ctg?gtg?aag?cct
49 ggg?gct?tca?gtg?aag?ata?tcc?tgc?aag?gct?tct?ggc?tat?acc?ttc?aca
97 agc?tac?tat?ata?cac?tgg?gtg?aag?cag?agg?cct?gga?cag?ggc?ctt?gag
145 tgg?att?gga?tat?att?tat?cct?aga?gat?ggt?agt?act?aat?tac?aat?gag
193 aag?ttc?aag?ggc?aag?gcc?aca?ctg?act?gca?gac?aca?tcc?tcc?agc?aca
241 gcc?tac?atg?cag?ctc?agc?agc?ctg?aca?tct?gag?gac?tct?gca?gtc?tat
289 ttc?tgt?gca?agg?gcc?ctc?tat?gat?ggt?cac?tac?gtg?ggg?gct?atg?gac
337 tac?tgg?ggt?caa?gga?gga?ggt?ggc?gga?tct?gga?ggg?ggt?ggt?agc?ggt
385 gga?ggc?ggg?agt?ggg?gcc?att?tcc?cag?gct?gtt?gtg?act?cag?gaa?tct
433 gca?ctc?acc?aca?tca?cct?ggt?gaa?aca?gtc?aca?ctc?act?tgt?cgc?tca
481 agt?act?ggg?gct?gtt?aca?acc?agt?aac?tat?gcc?aac?tgg?gtc?caa?gaa
529 aaa?cca?gat?cat?tta?ttc?act?ggt?cta?ata?ggt?ggt?acc?aac?aac?cga
577 gct?cca?ggt?gtt?cct?gcc?aga?atc?tca?ggc?tcc?ctg?att?gga?gac?aag
625 gct?gcc?ctc?acc?atc?aca?ggg?gct?cag?act?gag?gat?gag?gca?ata?tat
673 ttc?tgt?gtt?cta?tgg?tac?agc?aac?cat?tgg?gtg?ttc?ggt?gga?gga?acc
721 aaa?ctg。
The aminoacid sequence of the single-chain antibody of anti-human death receptor 5 (aDR5-scFv) gene is:
1 MET?Gly?Gln?Val?Gln?Leu?Gln?Gln?Ser?Gly?Pro?Glu?Leu?Val?Lys?Pro
17 Gly?Ala?Ser?Val?Lys?Ile?Ser?Cys?Lys?Ala?Ser?Gly?Tyr?Thr?Phe?Thr
33 Ser?Tyr?Tyr?Ile?His?Trp?Val?Lys?Gln?Arg?Pro?Gly?Gln?Gly?Leu?Glu
49 Trp?Ile?Gly?Tyr?Ile?Tyr?Pro?Arg?Asp?Gly?Ser?Thr?Asn?Tyr?Asn?Glu
65 Lys?Phe?Lys?Gly?Lys?Ala?Thr?Leu?Thr?Ala?Asp?Thr?Ser?Ser?Ser?Thr
81 Ala?Tyr?MET?Gln?Leu?Ser?Ser?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Val?Tyr
97 Phe?Cys?Ala?Arg?Ala?Leu?Tyr?Asp?Gly?His?Tyr?Val?Gly?Ala?MET?Asp
113 Tyr?Trp?Gly?Gln?Gly?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly
129 Gly?Gly?Gly?Ser?Gly?Ala?Ile?Ser?Gln?Ala?Val?Val?Thr?Gln?Glu?Ser
145 Ala?Leu?Thr?Thr?Ser?Pro?Gly?Glu?Thr?Val?Thr?Leu?Thr?Cys?Arg?Ser
161 Ser?Thr?Gly?Ala?Val?Thr?Thr?Ser?Asn?Tyr?Ala?Asn?Trp?Val?Gln?Glu
177 Lys?Pro?Asp?His?Leu?Phe?Thr?Gly?Leu?Ile?Gly?Gly?Thr?Asn?Asn?Arg
193 Ala?Pro?Gly?Val?Pro?Ala?Arg?Ile?Ser?Gly?Ser?Leu?Ile?Gly?Asp?Lys
209 Ala?Ala?Leu?Thr?Ile?Thr?Gly?Ala?Gln?Thr?Glu?Asp?Glu?Ala?Ile?Tyr
225 Phe?Cys?Val?Leu?Trp?Tyr?Ser?Asn?His?Trp?Val?Phe?Gly?Gly?Gly?Thr
241Lys?Leu?＊。
(*: terminator codon)
Claims (1)
1. the single-chain antibody gene of anti-human death receptor 5, its coding connects the variable region of heavy chain of antibody and the polypeptide of variable region of light chain by connection peptides, and said gene is by 726 based compositions;
Its nucleotides sequence is classified as:
1 atg?gga?cag?gtc?cag?ctg?cag?caa?tct?gga?cct?gag?ctg?gtg?aag?cct
49 ggg?gct?tca?gtg?aag?ata?tcc?tgc?aag?gct?tct?ggc?tat?acc?ttc?aca
97 agc?tac?tat?ata?cac?tgg?gtg?aag?cag?agg?cct?gga?cag?ggc?ctt?gag
145 tgg?att?gga?tat?att?tat?cct?aga?gat?ggt?agt?act?aat?tac?aat?gag
193 aag?ttc?aag?ggc?aag?gcc?aca?ctg?act?gca?gac?aca?tcc?tcc?agc?aca
241 gcc?tac?atg?cag?ctc?agc?agc?ctg?aca?tct?gag?gac?tct?gca?gtc?tat
289 ttc?tgt?gca?agg?gcc?ctc?tat?gat?ggt?cac?tac?gtg?ggg?gct?atg?gac
337 tac?tgg?ggt?caa?gga?gga?ggt?ggc?gga?tct?gga?ggg?ggt?ggt?agc?ggt
385 gga?ggc?ggg?agt?ggg?gcc?att?tcc?cag?gct?gtt?gtg?act?cag?gaa?tct
433 gca?ctc?acc?aca?tca?cct?ggt?gaa?aca?gtc?aca?ctc?act?tgt?cgc?tca
481 agt?act?ggg?gct?gtt?aca?acc?agt?aac?tat?gcc?aac?tgg?gtc?caa?gaa
529 aaa?cca?gat?cat?tta?ttc?act?ggt?cta?ata?ggt?ggt?acc?aac?aac?cga
577 gct?cca?ggt?gtt?cct?gcc?aga?atc?tca?ggc?tcc?ctg?att?gga?gac?aag
625 gct?gcc?ctc?acc?atc?aca?ggg?gct?cag?act?gag?gat?gag?gca?ata?tat
673 ttc?tgt?gtt?cta?tgg?tac?agc?aac?cat?tgg?gtg?ttc?ggt?gga?gga?acc
721 aaa?ctg；
The aminoacid sequence of its coded polypeptide is:
1 MET?Gly?Gln?Val?Gln?Leu?Gln?Gln?Ser?Gly?Pro?Glu?Leu?Val?Lys?Pro
17 Gly?Ala?Ser?Val?Lys?Ile?Ser?Cys?Lys?Ala?Ser?Gly?Tyr?Thr?Phe?Thr
33 Ser?Tyr?Tyr?Ile?His?Trp?Val?Lys?Gln?Arg?Pro?Gly?Gln?Gly?Leu?Glu
49 Trp?Ile?Gly?Tyr?Ile?Tyr?Pro?Arg?Asp?Gly?Ser?Thr?Asn?Tyr?Asn?Glu
65 Lys?Phe?Lys?Gly?Lys?Ala?Thr?Leu?Thr?Ala?Asp?Thr?Ser?Ser?Ser?Thr
81 Ala?Tyr?MET?Gln?Leu?Ser?Ser?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Val?Tyr
97 Phe?Cys?Ala?Arg?Ala?Leu?Tyr?Asp?Gly?His?Tyr?Val?Gly?Ala?MET?Asp
113 Tyr?Trp?Gly?Gln?Gly?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly
129 Gly?Gly?Gly?Ser?Gly?Ala?Ile?Ser?Gln?Ala?Val?Val?Thr?Gln?Glu?Ser
145 Ala?Leu?Thr?Thr?Ser?Pro?Gly?Glu?Thr?Val?Thr?Leu?Thr?Cys?Arg?Ser
161 Ser?Thr?Gly?Ala?Val?Thr?Thr?Ser?Asn?Tyr?Ala?Asn?Trp?Val?Gln?Glu
177 Lys?Pro?Asp?His?Leu?Phe?Thr?Gly?Leu?Ile?Gly?Gly?Thr?Asn?Asn?Arg
193 Ala?Pro?Gly?Val?Pro?Ala?Arg?Ile?Ser?Gly?Ser?Leu?Ile?Gly?Asp?Lys
209 Ala?Ala?Leu?Thr?Ile?Thr?Gly?Ala?Gln?Thr?Glu?Asp?Glu?Ala?Ile?Tyr
225 Phe?Cys?Val?Leu?Trp?Tyr?Ser?Asn?His?Trp?Val?Phe?Gly?Gly?Gly?Thr
241 Lys?Leu。
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| Application Number | Priority Date | Filing Date | Title |
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| CN2009101127988A CN101717775B (en) | 2009-11-13 | 2009-11-13 | Single-chain antibody gene of anti-human death receptor 5 |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009101127988A CN101717775B (en) | 2009-11-13 | 2009-11-13 | Single-chain antibody gene of anti-human death receptor 5 |
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| CN101717775A CN101717775A (en) | 2010-06-02 |
| CN101717775B true CN101717775B (en) | 2012-01-04 |
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| RU2748620C2 (en) * | 2015-07-16 | 2021-05-28 | Инхибркс, Инк. | Multivalent and multispecific hybrid proteins that bind to dr5 |
| CN106397594B (en) * | 2016-10-25 | 2019-06-04 | 中国药科大学 | A kind of agonist single-chain antibody of the anti-human death receptor 5 of full source of people and application |
| CN109957025A (en) * | 2017-12-25 | 2019-07-02 | 深圳宾德生物技术有限公司 | It is a kind of to target the single-chain antibody of DR5, Chimeric antigen receptor T cell and its preparation method and application |
| WO2020047705A1 (en) * | 2018-09-03 | 2020-03-12 | 安菲尼生命科技有限公司 | Dr5 single-domain antibody and use thereof |
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