CN101891818B - Single-chain antibody in anti-human prostate-specific membrane antigen extracellular region and application thereof - Google Patents
Single-chain antibody in anti-human prostate-specific membrane antigen extracellular region and application thereof Download PDFInfo
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- CN101891818B CN101891818B CN200910020936XA CN200910020936A CN101891818B CN 101891818 B CN101891818 B CN 101891818B CN 200910020936X A CN200910020936X A CN 200910020936XA CN 200910020936 A CN200910020936 A CN 200910020936A CN 101891818 B CN101891818 B CN 101891818B
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- chain antibody
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- specific membrane
- membrane antigen
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Abstract
The invention provides a single-chain antibody in an anti-human prostate-specific membrane antigen (PSMA) extracellular region, which adopts the phage display technology. The antibody is obtained by screening and enriching a large-storage capacity single-chain antibody library which is established by using peripheral blood mono-nuclear cells of a prostatic cancer patient through successive relaxation and rigorousness. The antibody can be solubly expressed in colon bacillus, and the affinity of the antibody with the prostate-specific membrane antigen extracellular region reaches Kd of 0.2nM. The antibody not only can be combined with the recombinant PSMA extracellular region, but also can be combined with NEK 293 cells with PSMA expressed on surface thereof and a prostate adenocarcinoma cell LNCaP, thus laying a foundation for adopting the antibody to perform prostate adenocarcinoma image diagnosis and guiding treatment.
Description
Technical field
The invention belongs to genetic engineering antibody, relate to the humanized's single-chain antibody and the application in prostate cancer diagnosis and medicine exploitation thereof of a kind of anti-human prostate-specific membrane antigen (PSMA) extracellular region.
Background technology
Prostate cancer is one of malignant tumour of current serious harm human health, and for its early diagnosis and therapy, especially the early diagnosis and therapy of transfer, recurrent foci is the difficult medical problem that people are difficult to capture.Constantly perfect along with tumor immunology and molecular biological continuous development, especially phage display technique is for the early diagnosis and the radical cure of prostate cancer brought new opportunity.At present, anti-prostate cancer antibody is carrying out clinical trial abroad, and these antibody all are humanized mouse endogenous antibody basically.Although humanization can reduce the immunogenicity of mouse endogenous antibody to a great extent, can not thoroughly remove its immunogenicity, thereby thereby in clinical treatment, can cause the appearance of neutralizing antibody to reduce its therapeutic value inevitably.Directly the single-chain antibody of screening high-affinity becomes the early diagnosis of prostate cancer, the important means of targeted therapy drug research undoubtedly from humanized's single-chain antibody library that patients with prostate cancer makes up.
Summary of the invention
The objective of the invention is to utilize phage antibody library technique to screen a kind of human prostate-specific membrane antigen (PSMA) extracellular region specificity humanized single-chain antibody.
Another object of the present invention is the application of above-mentioned single-chain antibody in prostatic cancer early diagnosis and targeted therapy.Utilize the expression level that this specificity humanized single-chain antibody can specific detection human prostate-specific membrane antigen (PSMA), as a standard of diagnosing prostate cancer; Utilize this human prostate-specific membrane antigen (PSMA) extracellular region specificity humanized single-chain antibody as bio-carrier simultaneously; Arrive prostate cancer cell in conjunction with the antitumor drug targeted of optimizing; Orientation is killed cancer cells, in treatment of prostate cancer, plays a significant role.
The present invention adopts phage display technique; Extract mRNA and therefrom amplify people's repertoire antibody variable region gene from the prostate cancer peripheral blood mononuclear cells, be assembled into the single-chain antibody gene fragment then and be cloned into the phasmid carrier and transformed into escherichia coli is built into humanized's single-chain antibody library of a big storage capacity.Use recombinant human PSMA (PSMA) extracellular region to screen then and obtain its specific high-affinity antibody.This antibody can be in intestinal bacteria solubility expression, the aminoacid sequence of this antibody is following:
L?P?V?L?T?Q?S?P?S?A?S?A?S?L?G?A?S?V?K?L?T?C T?L?S?S?W?H?S?S?N?A?I
A?W?H?Q?L?R?P?E?K?G?L?R?Y?L?M?K?V?N?S?D?G?S?H?N?W?G?D?G?I?P?D?R?F
S?G?S?S?S?G?A?E?R?Y?L?I?I?S?S?L?Q?S?E?D?E?A?D?Y?Y?C?Q?T?W?G?T?G?I?H
V?V?F?G?G?G?T?K?L?T?V?L?G?G?G?G?S?G?G?G?G?S?G?G?G?G?S?E?V?Q?L?L?Q
S?G?G?G?V?V?R?P?G?R?S?L?R?L?S?C?A?A?S?G?F?T?F?S N?Y?A?M?H?W?V?R
Q?A?P?G?K?G?L?E?W?V?A?V?M?S?Y?D?G?N?N?K?Y?Y?A?D?S?V?K?G?R?F?T?I?S
R?D?N?S?K?N?T?L?Y?L?Q?M?S?S?L?R?A?G?D?T?A?V?Y?Y?C?A?R?D?L?D?I?A?A
R?F?H?Y?C?A?M?D?V?W?G?Q?G?T?A?V?T?V?S?S
This aminoacid sequence is named as SEQ ID NO:1.
124 amino acid of heavy chain variable region gene coding of human prostate-specific membrane antigen of the present invention (PSMA) extracellular region specificity humanized single-chain antibody, sequence is following:
E?V?Q?L?L?Q?S?G?G?G?V?V?R?P?G?R?S?L?R?L?S?C?A?A?S?G?F?T?F?S N?Y?A
M?H?W?V?R?Q?A?P?G?K?G?L?E?W?V?A?V?M?S?Y?D?G?N?N?K?Y?Y?A?D?S?V?K
G?R?F?T?I?S?R?D?N?S?K?N?T?L?Y?L?Q?M?S?S?L?R?A?G?D?T?A?V?Y?Y?C?A?R
D?L?D?I?A?A?R?F?H?Y?C?A?M?D?V?W?G?Q?G?T?A?V?T?V?S?S
This aminoacid sequence is named as SEQ ID NO:2.
112 amino acid of chain variable region gene coding of human prostate-specific membrane antigen of the present invention (PSMA) extracellular region specificity humanized single-chain antibody, sequence is following:
L P?V?L?T?Q?S?P?S?A?S?A?S?L?G?A?S?V?K?L?T?C T?L?S?S?W?H?S?S?N?A?I
A?W?H?Q?L?R?P?E?K?G?L?R?Y?L?M?K?V?N?S?D?G?S?H?N?W?G?D?G?I?P?D?R?F
S?G?S?S?S?G?A?E?R?Y?L?I?I?S?S?L?Q?S?E?D?E?A?D?Y?Y?C?Q?T?W?G?T?G?I?H
V?V?F?G?G?G?T?K?L?T?V?L
This aminoacid sequence is named as SEQ ID NO:3.
Human prostate-specific membrane antigen of the present invention (PSMA) extracellular region specificity humanized single-chain antibody is made up of * 753 Nucleotide, and its sequence is following:
CTGCCTGTGCTGACTCAATCGCCCTCTGCCTCTGCCTCCCTGGGAGCCTCGGTCAAG
CTCACCTGCACTCTGAGCAGTTGGCACAGTAGCAACGCCATCGCATGGCATCAACT
GCGGCCAGAGAAGGGCCTTCGATATTTGATGAAAGTTAACAGTGATGGCAGCCACA
ACTGGGGAGACGGGATCCCTGATCGCTTCTCAGGCTCCAGCTCTGGGGCTGAGCGT
TACCTCATCATCTCCAGCCTCCAGTCTGAGGATGAGGCTGACTATTACTGTCAGACC
TGGGGCACTGGCATTCATGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGG
CGGTGGCGGATCTGGCGGAGGTGGCTCCGGAGGTGGCGGTTCTGAGGTGCAGCTGC
TGCAGTCTGGGGGAGGCGTGGTCCGGCCTGGGAGGTCCCTGAGACTCTCCTGTGCA
GCCTCTGGATTCACCTTCAGTAACTATGCTATGCACTGGGTCCGCCAGGCTCCAGGC
AAGGGGCTGGAGTGGGTGGCAGTTATGTCATATGATGGAAATAATAAATACTACGC
AGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGT
ATCTGCAAATGAGCAGCCTGCGAGCTGGGGACACGGCTGTATATTACTGTGCGAGA
GATCTGGATATAGCAGCTCGTTTCCACTACTGCGCTATGGACGTCTGGGGCCAAGG
GACTGCGGTCACCGTCTCCTCA
Human prostate-specific membrane antigen of the present invention (PSMA) extracellular region specificity humanized single-chain antibody variable region of heavy chain is made up of 372 Nucleotide, and its sequence is following:
GAGGTGCAGCTGCTGCAGTCTGGGGGAGGCGTGGTCCGGCCTGGGAGGTCCCTGAG
ACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACTATGCTATGCACTGGGTCCG
CCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATGTCATATGATGGAAATA
ATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCC
AAGAACACGCTGTATCTGCAAATGAGCAGCCTGCGAGCTGGGGACACGGCTGTATA
TTACTGTGCGAGA?GATCTGGATATAGCAGCTCGTTTCCACTACTGCGCTATGGACGT
CTGGGGCCAAGGGACTGCGGTCACCGTCTCCTCA
Human prostate-specific membrane antigen of the present invention (PSMA) extracellular region specificity humanized single-chain antibody variable region of heavy chain is by GHV3-30, the functional variable region gene with single ORF that forms after IGHD6-6 and the IGHJ6 rearrangement.Wherein the nucleotides sequence of CDR3 is classified GAT CTG GAT ATA GCA GCT CGT TTC as, and aminoacid sequence is D L D I A A R F, is made up of with the distinctive sequence of this antibody IGHD6-6.
Human prostate-specific membrane antigen of the present invention (PSMA) extracellular region specificity humanized single-chain antibody variable region of light chain is made up of 339 Nucleotide, and its sequence is following:
CTGCCTGTGCTGACTCAATCGCCCTCTGCCTCTGCCTCCCTGGGAGCCTCGGTCAAG
CTCACCTGCACTCTGAGCAGTTGGCACAGTAGCAACGCCATCGCATGGCATCAACT
GCGGCCAGAGAAGGGCCTTCGATATTTGATGAAAGTTAACAGTGATGGCAGCCACA
ACTGGGGAGACGGGATCCCTGATCGCTTCTCAGGCTCCAGCTCTGGGGCTGAGCGT
TACCTCATCATCTCCAGCCTCCAGTCTGAGGATGAGGCTGACTATTACTGTCAGACC
TGGGGCACTGGCATTCATGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGG
C
Human prostate-specific membrane antigen of the present invention (PSMA) extracellular region specificity humanized single-chain antibody variable region of light chain is reset the functional variable region gene with single ORF that the back forms by IGLV4-69 and IGLJ3 gene fragment.Wherein the nucleotides sequence of CDR3 is classified CAG ACC TGG GGC ACT GGC ATT as, and aminoacid sequence is: Q TW G T G I.
Humanized's single-chain antibody of anti-human prostate-specific membrane antigen of the present invention (PSMA) extracellular region can be used in prostate cancer diagnosis and medicine exploitation.
Phage antibody library technique need not through hybridoma technology, even need not can directly obtain specific high-affinity antibody molecule through immunologic process.Single-chain antibody has that molecular weight is little, and penetrance is strong, and the advantage that the transformation period is short is the diagnostic reagent of ideal tumor recurrence and metastasis, and also can treat for targeted drug provide guide effect.And will have long half time after the single-chain antibody that screens is transformed into whole antibody, can directly mediate complement and the effect of cellular immunization (ADCC) killing tumor cells.Therefore, the human prostate-specific membrane antigen among the present invention (PSMA) extracellular region specificity humanized single-chain antibody has significant values in the early diagnosis of prostate cancer and targeted therapy.
Human prostate-specific membrane antigen of the present invention (PSMA) extracellular region specificity humanized single-chain antibody can present expression at phage surface, also can be in intestinal bacteria solubility expression.Confirm that through protein electrophoresis and Western blot the soluble single-chain antibody molecule amount that IPTG induces the back to express is about 30kD.Show through ELISA and flow cytometry analysis: the avidity of this antibody and recombinant human PSMA (PSMA) extracellular region protein is Kd=0.2nM; This antibody not only can combine the PSMA extracellular region of recombinating, and can mating surface expresses NEK293 cell and the prostate cancer cell line LNCaP of PSMA.This is for utilizing this antibody and carry out the prostate cancer diagnostic imaging and targeted therapy being laid a good foundation.
Description of drawings
Fig. 1 is the purifying of human prostate-specific membrane antigen of the present invention (PSMA) extracellular region specificity humanized single-chain antibody;
Swimming lane 1:Marker; Swimming lane 2: stream is worn liquid; Swimming lane 3: first pass washing; 4: the second times washings of swimming lane; Swimming lane 5: the single-chain antibody of purifying.
Fig. 2 is that human prostate-specific membrane antigen of the present invention (PSMA) extracellular region specificity humanized's single-chain antibody and recombinant human PSMA extracellular region avidity are measured.
Fig. 3 is that human prostate-specific membrane antigen of the present invention (PSMA) extracellular region specificity humanized single-chain antibody combines active evaluation with the natural PS MA of cell surface expression;
A: only dye the NEK293 cell with anti-V5-APC; B: dye the NEK293 cell with single-chain antibody of the present invention and anti-V5-APC; C: only dye the NEK293 cell of PSMA transfection with anti-V5-APC; D: the NEK293 cell that dyes the PSMA transfection with single-chain antibody of the present invention and anti-V5-APC; E: only dye the LNCaP cell with anti-V5-APC; F: dye the LNCaP cell with single-chain antibody of the present invention and anti-V5-APC.
Embodiment
Embodiment in the face of human prostate-specific membrane antigen of the present invention (PSMA) extracellular region specificity humanized single-chain antibody is elaborated down.
Embodiment
(1) lymphocytic separation
The peripheral blood of collecting 50 patients with prostate cancer is total to 500ml, adds the centrifugal layering of isopyknic lymphocyte separation medium, 2500g, 10min.The careful middle level lymphocyte of drawing, centrifugal, 2500g, 10min.Abandon supernatant, with 1mlTrizol/5ml peripheral blood cracking lymphocyte, to solution thoroughly transparent till.-80 ℃ of preservations.
(2) extraction of RNA
The lymphocytolysis liquid of getting-70 ℃ of preservations is melted up to room temperature, adds 0.2ml chloroform/mlTrizol, the votex 15s that vibrates, and room temperature leaves standstill 3min, 12000g, 5min.Suct the limpid water of layer, note not sneaking into middle level albumen and DNA layer.Add 0.5ml Virahol/ml Trizol, put upside down mixing ,-20 ℃ of deposition 30-60min, the centrifugal 10min of 12000g.Keep deposition, 70% washing with alcohol one time is dried in the air, DEPC water dissolution precipitated rna.
(3) purifying of mRNA
Purification step is the conventional techniques of this area, is undertaken by (U.S. OMEGA) test kit specification sheets.
(4) reverse transcription
Get 13 μ g mRNA in 70 ℃ of insulation 10min, make it open secondary structure, place rapidly on ice then, by specification adds each reagent successively, and 6 aggressiveness carry out reverse transcription at random.Room temperature leaves standstill 10min and makes primer extension, and 42 ℃ of 60mi carry out reverse transcription then, last 95 ℃ of 5min deactivation ThermoScript II, ice bath 5min ,-20 ℃ of preservations.
(5) a complete set of variable region gene amplification
Reverse transcription product is divided into 3 parts carries out first round PCR, 3 ' the end primer of VH, VL, VK is mixed the back respectively hold each primer to increase respectively with 5 ' as template.Reaction conditions is: 94 ℃ of 5min warm starts, and 94 ℃ of 1min sex change, 55 ℃ of 1min annealing, 72 ℃ of 1min extend, altogether 35cycles.The last 7min that further extends.Reaction system is 100 μ l.The product VH of first round PCR is mixed, and VL and VK mixed with 1: 2, made the electrophoresis purifying and recovering respectively; After 100 times of dilutions as second take turns the PCR reaction template carry out second and take turns the PCR reaction and introduce longer protection sequence and overlapping extension sequence; Condition is: 94 ℃ of 5min warm starts, 94 ℃ of 1min sex change, 55 ℃ of 1min annealing; 72 ℃ of 1min extend, totally 25 circulations.Reaction system is 100 μ l.
(6) scFv (single-chain antibody) is pulsating splices at random and increases
With VH, VL and VK gene segment respectively the agarose gel electrophoresis of row 2% separate, reclaim; Then by VH: VK: VL: Llinker=3: 2: 1: 3 mixed is carried out overlapping extension with assembling scFv segment; Condition is 94 ℃ of 1min sex change; 55 ℃ of 1min annealing, 72 ℃ of 1min extend totally 7 circulations.With the dilution in capable 1: 100 of overlapping extension products, make template with this, the protection sequence of holding with VL 5 ' end and VH 3 ' is a primer amplification scFv segment; The PCR reaction conditions is 94 ℃ of 5min warm starts; 94 ℃ of 1min sex change, 65 ℃ of 1min annealing, 72 ℃ of 1min extend; After 10 circulations, carry out 15 circulations after again annealing temperature being dropped to 58 ℃.The PCR product is the single-chain antibody gene segment, and the sepharose with 1.5% reclaims.
(7) scFv and phasmid carrier is connected and the preparation of single-chain antibody library
1. the preparation of electric transformed competence colibacillus
Picking TG1 mono-clonal shakes bacterium and spends the night from the M9 flat board, transfers in 500ml LB jolting 2.5h, OD at 1: 100
600Value between 0.5-0.6, centrifugal receipts bacterium, 3000rpm, 10min.With the HEPES of isopyknic ice-cold 1mM washing twice, 3000rpm, 10min washs twice with ice-cold 10% glycerine of 1/10 volume, 4000rpm, 10min uses and precipitates the resuspended deposition of isopyknic ice-cold 10% glycerine, and every pipe 50 μ l are in-80 ℃ of preservations.Annotate: the competence preparation is all carried out at 0-4 ℃.
2. the preparation of single-chain antibody library
The single-chain antibody gene fragment of above-mentioned purifying is cut with Sfi I/Not I enzyme, and be connected with the phasmid carrier PCANTAB5E that cuts through same enzyme.Product be will connect and 10min deactivation T4DNA ligase enzyme, electric then transformed into escherichia coli TG1 hatched in 70 ℃.Bacterium after transforming is applied on the 2YT flat board that contains 1% glucose and 100mg/L ammonia benzyl, 30 ℃ of overnight cultures, the inferior daily 2YT that contains 15% glycerine scrapes off the clone frozen to-80 ℃ of preservations.
3. the phage surface of antibody library appears
(8) phage antibody library titer determination
Picking TG1 mono-clonal is in 5ml 2YT from the M9 substratum, and jolting is spent the night.Get the 0.5ml bacterium that spends the night and be inoculated among the 50ml 2YT (100ml Erlenmeyer flask), 37 ℃ of joltings are to OD600=0.2.Phage antibody library is done ten times of doubling dilutions, respectively get the TG1 that 10 μ l join 200 μ l OD600=0.2 mixing, 37 ℃ of water-bath 30min from each extent of dilution.The mixed solution of this 200 μ l bacterium and phage is applied on the 2TY agar plate that contains the ammonia benzyl.Inversion is put in 37 ℃, second day titre according to clone's number calculating phage library.
(9) preparation of complementary phage M13K07
Picking TG1 mono-clonal is in 5ml 2YT from the M9 substratum, and jolting is spent the night.Get the 0.5ml bacterium that spends the night and be inoculated among the 50ml2YT (100ml Erlenmeyer flask), 37 ℃ of joltings are to OD600=0.2.With ten times of doubling dilutions of M13K07, respectively get the TG1 that 10 μ l join 200 μ l OD600=0.2 mixing, 37 ℃ of water-bath 30min from each extent of dilution.The mixed solution of this 200 μ l bacterium and phage is joined in the top-layer agar glue of 3ml non-scald on hand, shake a little, pour into immediately on the agar plate with the 2TY preparation of 37 ℃ of preheatings.Inversion is put in 37 ℃, plaque can occur in second day.The single plaque of picking is inoculated among the TG1 of 3-4ml OD600=0.5 (preparation method is the same, promptly chooses the clone from M9 last evening, shakes bacterium and spends the night, and transfers in 50ml 2YT the about 2-2.5 of jolting hour in second day 1: 100), 37 ℃ of joltings 2 hours.The TG1 that this 3-4ml has been infected M13K07 is inoculated among the 500ml 2YT, 37 ℃ of joltings 1 hour.Add card that (50-70 μ g/ml), continue at 37 ℃ of jolting 8-16 hours, 10000rpm, centrifugal 15min receives supernatant.The PEG/NaCl (20% PEG6000, the NaCl of 2.5M) that adds 1/5 volume in the supernatant, mixing, 4 ℃ left standstill more than 1 hour, 10000rpm, centrifugal 15min.Contain the resuspended phage of PBS of 15% glycerine with 20ml, 10000rpm, centrifugal 15-20min collects supernatant (being precipitated as bacterial debris and PEG).Phage solution packing after the sterilization of 0.45 μ m membrane filtration, frozen in-80 ℃, survey its titre with the method for plaque simultaneously.
(10) screening of specific antibody
Antigen (first round) with 5ml 50ug/ml encapsulates the 25cm2 culturing bottle, and 4 ℃ are spent the night that (coating buffer is the sodium bicarbonate buffer liquid of 200mM, and PH9.6), second antigen concentration that encapsulate after taking turns is reduced to 5ml 10ug/ml.PBST washing back adds 5ml and contains 1 * 10
14The 4%MPBS of phage carries out the combination of antigen-antibody more than the room temperature water placing flat 90min behind the light rolling 30min on the decolorization swinging table.Respectively wash 20 times with PBS and PBST then, the 10ml TG1 that will grow to logarithmic phase at last adds culturing bottle and lets in 30 ℃ of water-bath 30min and is incorporated into antigenic phage-infect TG1.Infected TG1 coats on the 15cm 2YTGA flat board after centrifugal, and 30 ℃ are spent the night.Prepare phage with the method for complementary phage superingection from the TG1 that is infected and increased again, carry out second and take turns screening.Carrying out the screening process of " absorption-wash-out-infection-breeding " so repeatedly totally 4 takes turns.
The evaluation of (11) mono-clonal phage antibody antigen-binding activity
1. the preparation of mono-clonal phage antibody
Select single clone in 96 well culture plates that 2YTGA is housed from last plank of taking turns screening, 37 ℃ of joltings are spent the night, and transfer at 1: 100 next day in new 2YTGA; 37 ℃ of joltings 2.5 hours; Carry out superingection with M13K07 with 20: 1 ratio, 3500 change centrifugal 10min; With the resuspended bacterial precipitation of 200ul 2YTGAK, 30 ℃ of joltings are spent the night.Leave the heart 15min next day 11000, gets supernatant and carry out phage ELISA evaluation.
2.Phage it is active that ELISA identifies that antigen-antibody combines
Spending the night in 4 ℃ with PSMA encapsulates elisa plate, every hole 50ul, and concentration is 1ug/ml.The inferior daily PBS that contains 5% skim-milk fills it up with pore chamber temperature sealing 2 hours, and PBST washes 5 times, and with the PBS dilution antibody that contains 2% skim-milk, the antibody room temperature that dilution is good was placed 30 minutes.Wash plate with PBST and add antibody 200ul then 6 times, incubated at room 2 hours, PBST washes plate, 6 times.Add the antibody 200ul of the anti-M13 phage of HRP mark, extent of dilution is 1: 5000.Incubated at room 1 hour, PBST adds the TMB colour developing after washing plate 8 times.
The expression of (12) soluble single-chain antibody
Positive bacteriophage need infect HB2151 just can carry out solubility expression.(this step of filtering membrane is than HB2151 growth vigor to be arranged in order to remove the TG1 intestinal bacteria .TG1 that is mingled in the phage to the filter membrane of mistake 0.45 behind 10 or 100 times of doubling dilutions of phage work of positive colony; So remove it), get the HB2151 that dilution 10 μ l well remove to infect 200 μ l logarithmic phases.Method is following: grow in HB2151 mono-clonal on the M9 substratum in 5ml2YTG, transfer at 1: 100 next day in 50ml 2YTG, 37 ℃ of joltings are to OD600=0.5.Add 10 μ l through the phage filterable, that doubling dilution is crossed, mixing, 37 ℃ of water-bath 30min.The shop scribbles 2YTG agar plate how to decide ketone acid and ammonia benzyl, and 37 ℃ are spent the night.Choose mono-clonal in 5ml 2YTGA (containing 1% glucose), 37 ℃ are shaken and spend the night.Transfer at 1: 100 next day and contain among the 2YTGA of 0.1% glucose in 50ml; 37 ℃ of joltings are to OD600=0.9 (about 3 hours 40 minutes); Add IPTG to final concentration be 1mM; 30 ℃ of joltings are spent the night, and get supernatant and do capable polyacrylamide gel electrophoresis analysis expression, identify the antigen-binding activity of the single-chain antibody of solubility expression with ELISA.
(14) soluble single-chain purifying antibody
Because single-chain antibody is with the label amalgamation and expression of 6 Histidines; So the solubility expression supernatant can carry out affinity purification with the nickel post, concrete steps are: with PBS balance affinity column, sample upper prop; The imidazoles wash-out of 50mM is non-specific conjugated protein; Be incorporated into the single-chain antibody on the nickel post under the imidazoles wash-out of 500mM, remove imidazoles with molecular sieve, damping fluid is replaced into PBS.(see figure 1)
The avidity of (15) soluble single-chain antibody is identified
PSMA with 1ug/m l and two kinds of different concns of 0.5ug/ml encapsulates elisa plate; Add the single-chain antibody that the purifying of ten times of doubling dilutions from 1ug/ml to 1pg/ml is crossed then; Add anti-V5-HRP antibody (single-chain antibody and V5 label amalgamation and expression) then, show with TMB at last.Draw the avidity curve according to the ELISA reading; The corresponding AC of 1ug/ml and 0.5ug/ml PSMA when obtaining OD50 then; Be designated as [Ab] (corresponding to 1ug/ml PSMA) and [Ab] respectively ' (corresponding to 0.5ug/ml PSMA), use formula Kd=2 [Ab] '-[Ab] to calculate the avidity of single-chain antibody then.(see figure 2)
(16) soluble antibody combines active evaluation with the natural PS MA of cell surface expression
Because the conformation of reorganization PSMA possibly be different from the native conformation that is expressed in prostate cancer cell surface PSMA, combine active scFv possibly can not combine to be expressed in the PSMA of cell surface so have with reorganization PSMA.Thereby single-chain antibody and the natural activity that combines that is expressed in cell surface PSMA in order to identify that we screen; We with Flow Cytometry to this antibody and NEK293 cell, transfection NEK293 cell and the combination activity of prostate cancer cell line LNCaP of PSMA compare.Method is that this antibody and above-mentioned three kinds of cells were hatched 1 hour, and control group does not add this antibody, uses anti-V5-APC antibody staining then, compares the power of apc signal at last with flow cytometer.(seeing Fig. 3 A-F)
Need to prove that accompanying drawing 1 provided by the invention is the picture of under the gel imaging appearance, gathering, Fig. 3 A-F is that flow cytometer detects the picture that the back generates automatically, is medical research picture the most clearly.
Be not described in detail the known technology that part belongs to the industry or relevant industries among the above embodiment, the equipment of employing is industrial practice equipment.
Sequence table
1、SEQ?ID：1。
Sequence:
L?P?V?L?T?Q?S?P?S?A?S?A?S?L?G?A?S?V?K?L?T?C T?L?S?S?W?H?S?S?N?A?I
A?W?H?Q?L?R?P?E?K?G?L?R?Y?L?M?K?V?N?S?D?G?S?H?N?W?G?D?G?I?P?D?R?F
S?G?S?S?S?G?A?E?R?Y?L?I?I?S?S?L?Q?S?E?D?E?A?D?Y?Y?C?Q?T?W?G?T?G?I?H
V?V?F?G?G?G?T?K?L?T?V?L?G?G?G?G?S?G?G?G?G?S?G?G?G?G?S?E?V?Q?L?L?Q
S?G?G?G?V?V?R?P?G?R?S?L?R?L?S?C?A?A?S?G?F?T?F?S N?Y?A?M?H?W?V?R
Q?A?P?G?K?G?L?E?W?V?A?V?M?S?Y?D?G?N?N?K?Y?Y?A?D?S?V?K?G?R?F?T?I?S
R?D?N?S?K?N?T?L?Y?L?Q?M?S?S?L?R?A?G?D?T?A?V?Y?Y?C?A?R?D?L?D?I?A?A
R?F?H?Y?C?A?M?D?V?W?G?Q?G?T?A?V?T?V?S?S
Be made up of 753 Nucleotide, its sequence is following:
CTGCCTGTGCTGACTCAATCGCCCTCTGCCTCTGCCTCCCTGGGAGCCTCGGTCAAG
CTCACCTGCACTCTGAGCAGTTGGCACAGTAGCAACGCCATCGCATGGCATCAACT
GCGGCCAGAGAAGGGCCTTCGATATTTGATGAAAGTTAACAGTGATGGCAGCCACA
ACTGGGGAGACGGGATCCCTGATCGCTTCTCAGGCTCCAGCTCTGGGGCTGAGCGT
TACCTCATCATCTCCAGCCTCCAGTCTGAGGATGAGGCTGACTATTACTGTCAGACC
TGGGGCACTGGCATTCATGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGG
CGGTGGCGGATCTGGCGGAGGTGGCTCCGGAGGTGGCGGTTCTGAGGTGCAGCTGC
TGCAGTCTGGGGGAGGCGTGGTCCGGCCTGGGAGGTCCCTGAGACTCTCCTGTGCA
GCCTCTGGATTCACCTTCAGTAACTATGCTATGCACTGGGTCCGCCAGGCTCCAGGC
AAGGGGCTGGAGTGGGTGGCAGTTATGTCATATGATGGAAATAATAAATACTACGC
AGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGT
ATCTGCAAATGAGCAGCCTGCGAGCTGGGGACACGGCTGTATATTACTGTGCGAGA
GATCTGGATATAGCAGCTCGTTTCCACTACTGCGCTATGGACGTCTGGGGCCAAGG
GACTGCGGTCACCGTCTCCTCA
2、SEQ?ID?NO：2。
Sequence:
E?V?Q?L?L?Q?S?G?G?G?V?V?R?P?G?R?S?L?R?L?S?C?A?A?S?G?F?T?F?S N?Y?A
M?H?W?V?R?Q?A?P?G?K?G?L?E?W?V?A?V?M?S?Y?D?G?N?N?K?Y?Y?A?D?S?V?K
G?R?F?T?I?S?R?DN?S?K?N?T?L?Y?L?Q?M?S?S?L?R?A?G?D?T?A?V?Y?Y?C?A?R
D?L?D?I?A?A?R?F?H?Y?C?A?M?D?V?W?G?Q?G?T?A?V?T?V?S?S
Be made up of 372 Nucleotide, its sequence is following:
GAGGTGCAGCTGCTGCAGTCTGGGGGAGGCGTGGTCCGGCCTGGGAGGTCCCTGAG
ACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACTATGCTATGCACTGGGTCCG
CCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATGTCATATGATGGAAATA
ATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCC
AAGAACACGCTGTATCTGCAAATGAGCAGCCTGCGAGCTGGGGACACGGCTGTATA
TTACTGTGCGAGAGATCTGGATATAGCAGCTCGTTTCCACTACTGCGCTATGGACGT
CTGGGGCCAAGGGACTGCGGTCACCGTCTCCTCA
Human prostate-specific membrane antigen of the present invention (PSMA) extracellular region specificity humanized single-chain antibody variable region of heavy chain is by GHV3-30, the functional variable region gene with single ORF that forms after IGHD6-6 and the IGHJ6 rearrangement.Wherein the nucleotides sequence of CDR3 is classified GAT CTG GAT ATA GCA GCT CGT TTC as, and aminoacid sequence is D L D I A A R F, is made up of with the distinctive sequence of this antibody IGHD6-6.
3、SEQ?ID?NO：3
Sequence:
L?P?V?L?T?Q?S?P?S?A?S?A?S?L?G?A?S?V?K?L?T?C T?L?S?S?W?H?S?S?N?A?I
A?W?H?Q?L?R?P?E?K?G?L?R?Y?L?M?K?V?N?S?D?G?S?H?N?W?G?D?G?I?P?D?R?F
S?G?S?S?S?G?A?E?R?Y?L?I?I?S?S?L?Q?S?E?D?E?A?D?Y?Y?C?Q?T?W?G?T?G?I?H
V?V?F?G?G?G?T?K?L?T?V?L
Be made up of 339 Nucleotide, its sequence is following:
CTGCCTGTGCTGACTCAATCGCCCTCTGCCTCTGCCTCCCTGGGAGCCTCGGTCAAG
CTCACCTGCACTCTGAGCAGTTGGCACAGTAGCAACGCCATCGCATGGCATCAACT
GCGGCCAGAGAAGGGCCTTCGATATTTGATGAAAGTTAACAGTGATGGCAGCCACA
ACTGGGGAGACGGGATCCCTGATCGCTTCTCAGGCTCCAGCTCTGGGGCTGAGCGT
TACCTCATCATCTCCAGCCTCCAGTCTGAGGATGAGGCTGACTATTACTGTCAGACC
TGGGGCACTGGCATTCATGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGG
C
Human prostate-specific membrane antigen of the present invention (PSMA) extracellular region specificity humanized single-chain antibody variable region of light chain is reset the functional variable region gene with single ORF that the back forms by IGLV4-69 and IGLJ3 gene fragment.Wherein the nucleotides sequence of CDR3 is classified CAG ACC TGG GGC ACT GGC ATT as, and aminoacid sequence is: Q TW G T G I.
Claims (5)
1. humanized's single-chain antibody of an anti-human prostate-specific membrane antigen extracellular region, it is characterized in that: the aminoacid sequence of this single-chain antibody is SEQ ID NO:1
The aminoacid sequence of this antibody is following:
L P V L T Q S P S A S A S L G A S V K L T C T L S S
W H S S N A I A W H Q L R P E K G L R Y L M K V N S
D G S H N W G D G I P D R F S G S S S G A E R Y L I
I S S L Q S E D E A D Y Y C Q T W G T G I H V V F G
G G T K L T V L G G G G S G G G G S G G G G S E V Q
L L Q S G G G V V R P G R S L R L S C A A S G F T F
S N Y A M H W V R Q A P G K G L E W V A V M S Y D
G N N K Y Y A D S V K G R F T I S R D N S K N T L Y
L Q M S S L R A G D T A V Y Y C A R D L D I A A R F
H Y C A M D V W G Q G T A V T V S S。
2. humanized's single-chain antibody of anti-human prostate-specific membrane antigen extracellular region according to claim 1, it is characterized in that: said humanized's single-chain antibody is made up of 753 Nucleotide, and its sequence is following:
CTGCCTGTGCTGACTCAATCGCCCTCTGCCTCTGCCTCCCTGGGAGCCTCGGTCAAGCTCACCTGCACTCTGAGCAGTTGGCACAGTAGCAACGCCATCGCATGGCATCAACTGCGGCCAGAGAAGGGCCTTCGATATTTGATGAAAGTTAACAGTGATGGCAGCCACAACTGGGGAGACGGGATCCCTGATCGCTTCTCAGGCTCCAGCTCTGGGGCTGAGCGTTACCTCATCATCTCCAGCCTCCAGTCTGAGGATGAGGCTGACTATTACTGTCAGACCTGGGGCACTGGCATTCATGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGCGGTGGCGGATCTGGCGGAGGTGGCTCCGGAGGTGGCGGTTCTGAGGTGCAGCTGCTGCAGTCTGGGGGAGGCGTGGTCCGGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATGTCATATGATGGAAATAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAGCAGCCTGCGAGCTGGGGACACGGCTGTATATTACTGTGCGAGAGATCTGGATATAGCAGCTCGTTTCCACTACTGCGCTATGGACGTCTGGGGCCAAGGGACTGCGGTCACCGTCTCCTCA。
3. humanized's single-chain antibody of anti-human prostate-specific membrane antigen extracellular region according to claim 1; It is characterized in that: said human prostate-specific membrane antigen extracellular region specificity humanized single-chain antibody variable region of heavy chain is made up of 372 Nucleotide, and its sequence is following:
GAGGTGCAGCTGCTGCAGTCTGGGGGAGGCGTGGTCCGGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATGTCATATGATGGAAATAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAGCAGCCTGCGAGCTGGGGACACGGCTGTATATTACTGTGCGAGAGATCTGGATATAGCAGCTCGTTTCCACTACTGCGCTATGGACGTCTGGGGCCAAGGGACTGCGGTCACCGTCTCCTCA。
4. humanized's single-chain antibody of anti-human prostate-specific membrane antigen extracellular region according to claim 1; It is characterized in that: said human prostate-specific membrane antigen extracellular region specificity humanized single-chain antibody variable region of light chain is made up of 339 Nucleotide, and its sequence is following:
CTGCCTGTGCTGACTCAATCGCCCTCTGCCTCTGCCTCCCTGGGAGCCTCGGTCAAGCTCACCTGCACTCTGAGCAGTTGGCACAGTAGCAACGCCATCGCATGGCATCAACTGCGGCCAGAGAAGGGCCTTCGATATTTGATGAAAGTTAACAGTGATGGCAGCCACAACTGGGGAGACGGGATCCCTGATCGCTTCTCAGGCTCCAGCTCTGGGGCTGAGCGTTACCTCATCATCTCCAGCCTCCAGTCTGAGGATGAGGCTGACTATTACTGTCAGACCTGGGGCACTGGCATTCATGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGC。
5. humanized's single-chain antibody of anti-human prostate-specific membrane antigen extracellular region according to claim 1 is used for treating the application of the medicine of prostate cancer in preparation.
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| CN105504061A (en) * | 2016-02-03 | 2016-04-20 | 南昌大学 | Nano-antibody directing at prostate-specific membrane antigen |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105968203A (en) * | 2016-02-03 | 2016-09-28 | 南昌大学 | Single-domain heavy chain antibody for anti-prostate specific membrane antigen extracellular region |
| CN105968202A (en) * | 2016-02-03 | 2016-09-28 | 南昌大学 | Single-domain heavy-chain antibody aiming at prostate specific membrane antigen extracellular region |
| CN105968201A (en) * | 2016-02-03 | 2016-09-28 | 南昌大学 | Single-domain heavy-chain antibody aiming at prostate specific membrane antigen |
| CN105524171A (en) * | 2016-02-03 | 2016-04-27 | 中国人民解放军第三军医大学第三附属医院 | VHH (variable domain of heavy chain of heavy-chain) antibody for prostate specific membrane antigen |
| CN105622755B (en) * | 2016-02-03 | 2019-09-06 | 中国人民解放军第三军医大学第一附属医院 | A kind of nano antibody of anti-prostate-specific membrane antigen extracellular region |
| GB201614162D0 (en) * | 2016-08-18 | 2016-10-05 | Polytherics Ltd | Antibodies, uses thereof and conjugates thereof |
| CN110407939B (en) * | 2019-03-12 | 2023-09-29 | 广东医科大学附属第二医院 | Humanized anti-PSMA single-chain antibody and application thereof |
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| CN1387539A (en) * | 1999-07-29 | 2002-12-25 | 米德列斯公司 | Human monoclonal antibodies to prostate specific membrane antigen |
| CN1652821A (en) * | 2002-01-28 | 2005-08-10 | 米德列斯公司 | Human monoclonal antibodies to prostate specific membrane antigen (PSMA) |
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| CN105504061A (en) * | 2016-02-03 | 2016-04-20 | 南昌大学 | Nano-antibody directing at prostate-specific membrane antigen |
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