CN105968205A - Nano antibody for anti-prostate specific membrane antigen - Google Patents
Nano antibody for anti-prostate specific membrane antigen Download PDFInfo
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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Abstract
The invention relates to a single-domain heavy chain antibody aiming at anti-prostate specific membrane antigen, which belongs to the field of gene engineering. The antibody has the protein with the amino acid sequence disclosed as SEQ ID NO:1, and can be used in the fields of immune detection, antigen enrichment purification and the like. The amino acid sequence provided by the invention can be used as a precursor, can be modified by using a random or site-directed mutagenesis technique to obtain a mutant with better properties (affinity, specificity, stability and the like), and is used for developing proteins or polypeptides which are further used in medicine, industry and agriculture.
Description
Technical field
The present invention relates to single domain heavy chain antibody technology (also known as nano antibody technology), and genetic engineering antibody technology, special
It not the single domain heavy chain antibody for prostate specific membrane antigen or polypeptide.
Technical background
Single domain antibody refers to the genetic engineering antibody being made up of common antibody variable region (VH or VL).Single domain heavy chain antibody
(it is also called nano antibody, VHH antibody, variable domain of heavy chain of heavy-chain
Antibody) refer to only to be made up of heavy chain antibody (heavy-chain antibody) variable region (Variable region)
Genetic engineering antibody, wherein, heavy chain antibody (heavy-chain antibody) is that one is present in the animal body such as camel, shark
The antibody of interior natural deletions light chain.Single domain heavy chain antibody is the minimum intact antigen binding fragment being currently known, and has molecule
Measure the features such as little, good penetrability, be widely used in the field such as basic research, medical diagnosis and detection, antibody drug exploitation at present.
Carcinoma of prostate is the malignant tumor that a kind of serious threat elderly men is healthy, and its early diagnosis and therapy is pre-for it
After have great importance.Prostate specific membrane antigen (prostate specific membrane antigen, PSMA)
It is a kind of II type transmembrane glycoprotein being positioned on prostatic cell film, is made up of 750 aminoacid, is divided into intracellular region (aminoacid
Sequence is 1-18), cross-film district (19-43) and extracellular region (44-750), relative to the prostate-specific being conventionally used to Clinical detection
Antigen (prostate specific antigen, PSA), is a kind of more sensitive and special prostate cancer label,
Especially in hormone-refractory prostate cancer and carcinoma of prostate metastasis, it is high expressed, distinguishes carcinoma of prostate and other classes
The sensitivity of type malignant tumor and specificity are respectively 65.9% and 94.5%.It addition, at the entity in multiple non-prostate source
Tumor, such as pulmonary carcinoma, bladder cancer, gastric cancer, cancer of pancreas, renal carcinoma and colorectal cancer etc., is expressed in tumor vessel PSMA also high special
On endotheliocyte.And itself there is neural carboxypeptidase and the activity of folic acid hydrolytic enzyme, the born of the same parents of 707 aminoacid compositions in addition
Outskirt can provide the features such as multiple epitopes so that it is becomes in tumour immunity targeted therapy and molecular imaging important
Research target spot.
At present, it has been found that multiple being also prepared for for PSMA multiple can occur specific binding material, bag with it
Including monoclonal antibody 7E11, J591, fit A9 and A10, ScFv antibody D7, Fab antibody and the humanization that recombinant technique obtains resists
The report of body, has passed through the U.S. FDA approval diagnosis for carcinoma of prostate and the 111In-spray of radionuclide therapy in late period
Ground peptide, it is simply that be connected with radionuclide based on the monoclonal antibody for PSMA and form.But compared with single domain heavy chain antibody, this
The shortcomings such as it is of a relatively high that a little parts exist production cost, and preparation process is complicated.
Summary of the invention
It is an object of the invention to provide the single domain heavy chain antibody for prostate specific membrane antigen, preparation can be used for
The reagent of detection prostate specific membrane antigen and instrument.
The present invention provides a single domain heavy chain antibody for prostate specific membrane antigen (i.e. a kind of anti-prostatitis of the present invention
The nano antibody of gland specific membrane antigen), there is the aminoacid sequence shown in SEQ ID NO.:1, its aminoacid sequence can pass through
Standardized antibody amino acids sequence method for numbering serial (ImMunoGeneTics, IMGT) is numbered the division with domain.
The present invention provides a kind of protein or polypeptide, it is characterized in that one or two the above ammonia comprising in framework region
Base acid sequence, and at least with an aminoacid sequence, there is 90% homology.
The present invention provides a kind of protein or polypeptide, it is characterized in that comprising in complementary determining region one or two more than
Aminoacid sequence, and at least with an aminoacid sequence, there is 80% homology.
The present invention provides a nucleic acid molecules, it is characterized in that encoding SEQ ID NO.:1, can be with by genetic codon
Time obtain the particular sequence of this nucleic acid molecules.The sequence of this nucleic acid molecules such as SEQ ID NO.:2.
The present invention also provides for a nucleic acid molecules, it is characterized in that encoding SEQ ID NO.:1 partial domain, by heredity
Codon can obtain the particular sequence of this nucleic acid molecules at any time.
Nucleotide sequence provided by the present invention or at least partly sequence can carry out table by suitable expression system
Reach to obtain corresponding protein or polypeptide.These expression systems include antibacterial, yeast, filamentous fungi, zooblast, insecticide
Cell, plant cell, or Cell free expression system.
The present invention also provides for a kind of carrier, comprises described nucleotide sequence.Owing to genetic codon has degeneracy, this nucleic acid
Sequence can be different according to different application purposes.
The present invention also provides for a kind of host cell, including described protein or expression vector.
The present invention also provides for a kind of detecting the method for prostate specific membrane antigen on cell, it is characterized in that containing above-mentioned egg
White matter or polypeptide.The protein provided based on the present invention or the polypeptide ability specific binding with prostate specific membrane antigen,
Set up the detection method of prostate specific membrane antigen.Wherein, preferred method includes enzyme linked immunosorbent assay (Enzyme-
Linked immunosorbent assay, ELISA), fluorescent immune method (Fluoroimmunoassay, FIA), immuno-chip
Method, affinity chromatography and immunochromatographic method.
The present invention is directed to prostate specific membrane antigen single domain heavy chain antibody non-diseases diagnoses and treatment purpose immune detection,
And the application in thalline or antigen enrichment purification.
Aminoacid sequence provided by the present invention can be transformed by random or site-directed mutagenesis technique as precursor,
It is obtained in that character (water solublity, stability, affinity and specificity etc.) preferably mutant, is used for developing being further used for
Medical, industrial, agriculture protein or polypeptide.
The invention still further relates to a kind of affine adsorbing material of the immunity for prostate specific membrane antigen, including carrier, take
The aglucon being loaded on carrier, this material using the single domain heavy chain antibody for prostate specific membrane antigen as aglucon, described pin
The single domain heavy chain antibody of prostate specific membrane antigen had the aminoacid sequence shown in SEQ ID NO.:1.Described carrier is
Magnetic bead, agarose gel microsphere, silica gel microball or porous material.
Some terms that the present invention is described have a following implication:
Homology: describe the similarity degree of two or more aminoacid sequences, first aminoacid sequence and second ammonia
Between base acid sequence, the percentage ratio of homology can be by [corresponding to second aminoacid sequence in first aminoacid sequence
The quantity of the amino acid residue that the aminoacid sequence of position is identical] exist divided by [aminoacid sum in first aminoacid sequence]
Be multiplied by [100%] to calculate, wherein certain the amino acid whose disappearance in second aminoacid sequence, insert, replace or add (with
First aminoacid is compared) it is considered as to have difference.Alternatively, percent homology can also utilize known for sequence
The Computing program such as NCBI Blast joined obtains.
Domain: the fundamental structural unit of tertiary protein structure, is generally of certain function.
IMGT numbers: the one in IMGT data base (The International ImMunoGeneTics Datbase)
The most standardized antibody amino acids sequence method for numbering serial.Concrete method for numbering serial be referred to document (Ehrenman, F.,
Q.Kaas, et al. (2010). " IMGT/3D structure-DB and IMGT/DomainGapAlign:a
Databaseand a tool for immunoglobulins or antibodies, T cell receptors, MHC,
IgSF and MhcSF. " Nucleic Acids Res38 (Database issue): D301-307.Lefranc, M.P.,
C.Pommie, et al. (2003). " IMGT unique numbering for immunoglobulin and T cell
receptor variable domains and Igsuperfamily V-like domains“Dev comp Immunol
Description in 27 (1): 55-77.http: //www.imgt.org/).
Codon (codon): be also called three disjunctor codons (triplet code), refer to corresponding to certain amino acid whose core
Thuja acid triplet.The position of polypeptide chain in this kind of aminoacid insertion growth is determined during translation.
Beneficial effects of the present invention: the single domain heavy chain antibody or the polypeptide that the present invention is directed to prostate specific membrane antigen have
Specific binding with prostate specific membrane antigen, can pass through biological method large-scale production, low cost, efficiently, molecule
Measure the character such as little, good penetrability, demonstrate good application prospect.
Accompanying drawing explanation
Fig. 1 bacterium colony PCR primer electrophoresis, recombiant protein electrophoresis and Western Blot identify figure.Left side Marker swimming lane is
DNA molecular amount standard, the bacterium colony PCR fragment in swimming lane 1 occurs in desired location.Centre carries out SDS-for albumen after purification
PAGE detects, and bright band occurs in desired location.The right is with anti-PSMA monoclonal antibody and anti-His tag antibody
Western Blot identifies figure.
Detailed description of the invention
Below by single domain heavy chain antibody (polypeptide) preparation, analyze and apply, the present invention will be further described, these
Specific embodiment is not construed in any way as limiting the range of application of the present invention.
Embodiment 1
The eukaryotic expression of prostate specific membrane antigen film outskirt
Use RNAiso Plus reagent to extract total serum IgE in the LNCaP cell of high expressed PSMA, utilize RT-PCR method to obtain
To the DNA fragmentation of coding PSMA extracellular domain fragment, Not I and two kinds of restricted enzyme of BamH I are used to be inserted into eucaryon table
Reach carrier pRAG2a, be connected as recombiant plasmid by T4DNA ligase.Recombiant plasmid thermal shock is transformed into TOP10 competent cell
Overnight incubation, will identify that correct clone send sequence verification.Extract the plasmid of positive colony, use liposome
Lipofectamine 2000, by cultivating in DNA plasmid transfection to HEK-293 cell, collects supernatant in different time phases, carries out
SDS-PAGE electrophoresis detection.After cultivating certain time, operate this albumen of purification according to HisTrap FF Crude test kit.By pure
After albumen after change carries out SDS-PAGE, electricity goes on pvdf membrane, 5% defatted milk powder close after, be separately added into PSMA antibody and
His antibody, 4 DEG C overnight；After rinsing, the anti-incubated at room temperature 1h that adds two, again rinse, add nitrite ion and carry out develop (Fig. 1).
Embodiment 2
Anti-prostate specific membrane antigen single domain heavy chain antibody (i.e. resists for anti-prostate specific membrane antigen single domain heavy chain
Body) elutriation and qualification
The method of solid phase elutriation is used to be coated with chase after from camel source natural antibody phage display library (for list of references: ", Xu Yang,
Liu Xia, etc. the structure in camel natural single domain heavy chain antibody storehouse, source and qualification [J]. Chinese biological engineering magazine, 2011,31 (4): 31-
36. " display libraries built in) in elutriation for the single domain heavy chain antibody of prostate specific membrane antigen.Prostate specific
The expression of membrane antigen film outskirt is carried out according to the above embodiments 1, during first round elutriation, employing phosphate buffered solution (PBS,
PH7.4) (2-4 wheel is coated concentration and is respectively 100,50,50 μ to 150 μ g/mL to dilute the PSMA extracellular region protein of above-mentioned synthesis
G/mL), in ELISA Plate, every hole adds 100 μ L, and 4 DEG C are coated overnight.PBST (containing 0.5%Tween 20) washes plate 5 times, and 3%
BSA-PBS closes 2h at 37 DEG C, washes plate 3 times, adds the phage antibody library 100 μ L hatched with 0.5%BSA-PBS (containing about 2
×1011CFU), hatch 1h for 37 DEG C, wash plate 5 times (increasing by 3 times by wheel) with PBST, then wash plate 10 times (increasing by 5 times by wheel) with PBS.
Again with the phage (37 DEG C, 5min) of 100 μ L eluents (glycine-HCI, pH 2.2) eluting absorption, with 50 μ L Tris-
HCl (1mol/L, pH 9.0) neutralizes eluate, takes 10 μ L for titer determination, washes in a pan for next round after remaining eluate amplification
Choosing.
The PSMA extracellular region protein coated elisa plate using concentration to be 5 μ g/mL after four-wheel elutriation, washes plate, closes together
On.The phage clone 37 DEG C adding amplification purification hatches 15min, and 1h is hatched for 37 DEG C in 100 μ L/ holes.Dilution factor is added after washing plate
It is the HRP-anti-M13 antibody of 1: 5000,100 μ L/ holes, hatch 1h for 37 DEG C.PBST washes plate 5 times, adds TMB working solution 100 μ L/
Hole, room temperature 20min, every hole adds 50 μ L sulphuric acid (concentration is 2mol/L) and terminates reaction, measures 450nm light absorption value.With previous round
The phage antibody library direct coated ELISA Plate of amplification, as positive control, with PBS replacement phage clone as blank, is used
Phage-ELISA method measures the combination activity of phage particle indirectly.
The indirect phage-ELISA of table 1 is loaded table
Send order-checking company to carry out sequencing ELISA positive colony, obtain anti-prostate specific membrane antigen single domain weight
The Insert Fragment DNA sequence of chain antibody phage positive colony, is specially (SEQ ID NO.:2):
ATGGCCCAGGTGCAGCTGGTGGAGTCGGGGGGGGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCC
TGTGCAGCCTCTGGACGCACCAAAAGTACCAGTTCTATGGGCTGGTTCCGCCAGGCTCCAGGAAAGGGGCGTGACTT
TGTAGCAGGTATTAGTGGTGCAGGTAAAACAAACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACG
GCGCCAAGAACACGGTGCATCTGCAAATGAACAGCATGAAACCTGAGGACACGGCCGTCTATTACTGTAACACCCTG
ATTCGACCCCGGGGGTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAGCGGC
CGC
The aminoacid sequence of its coding is as shown in SEQ ID NO.:1:
QVQLVESGGGLVQAGGSLRLSCAASGRTKSTSSMGWFRQAPGKGRDFVAGISGAGKTNYADSVKGRFTISRDGAKNT
VHLQMNSMKPEDTAVYYCNTLIRPRGWGQGTQVTVSS.The i.e. single domain heavy chain of the anti-prostate specific membrane antigen of the present invention
Antibody, it can occur specific binding with the film outskirt of psma protein.
Embodiment 3
The ELISA of PSMA express cell and fluorescence immunoassay detection
Gastric cancer cell MKN45 does not express PSMA, and prostate gland cancer cell LNCaP expresses PSMA, as a example by both cells,
The anti-prostate specific membrane antigen single domain heavy chain antibody phage positive colony that in embodiment 2, elutriation obtains is used to carry out cell
The ELISA of level and fluorescence immunoassay detection.Plant respectively in 96 orifice plates into LNCaP cell (expressing PSMA) and MKN45 cell is (no
Express PSMA) each 1 × 104Individual, overnight incubation.4% paraformaldehyde is fixed, and every hole drips the 3% hydrogen peroxide liquid of 100 μ L, uses
To block endogenous peroxidase activity, hatch 30min for 37 DEG C.TBS washes plate 3 times, and 5%BSA-PBS closes, and adds 100 μ L
Anti-prostate specific membrane antigen single domain heavy chain antibody phage positive colony, hatches 1h in 37 DEG C.Thereafter PBS rinsing, adds
HRP-anti-M13 antibody, TMB working solution and OD450Mensuration with embodiment 2.Positive control uses phage to substitute cell, empty
White comparison uses PBS to substitute the phage clone added, and is repeated 3 times.
Plant into LNCaP cell and MKN45 cell each 4 × 10 after cell climbing sheet is added in every hole in 24 orifice plates5Individual, training
Support overnight.Taking out creep plate, 4% paraformaldehyde fixes, rinsing, drips 3% hydrogen peroxide on creep plate surface, in order to block endogenous
Peroxidase activity.Thereafter TBS washes 3 times, and 5%BSA-PBS closes 30min.Drip 100 μ L and show that nanometer of the present invention resists
The phage clone of body hatches 1h, be not added with phage clone cell climbing sheet as blank.Thereafter adding dilution factor is 1:
The anti-M13 monoclonal antibody of 2000 hatches 30min.Drip the FITC-goat that 30 μ l dilution factors are 1: 200 after developing a film and resist little
Mus two resists, and lucifuge hatches 30min, and redyes with DAPI dyeing liquor, is placed in fluorescence microscopy Microscopic observation.
Result shows: there are differences with blank statistically owing to not expressing the MKN45 cell measured value of PSMA, table
This phage bright can occur non-specific binding with it；But LNCaP cell measured value is significantly higher than MKN45 cell, shows this
A part of difference comes from PSMA and the specificity of anti-prostate specific membrane antigen single domain heavy chain antibody phage positive colony
In conjunction with.Meanwhile, cellular immunofluorescence shows that LNCaP cell can be with the single domain heavy chain antibody for prostate specific membrane antigen
Positive colony combines, and MKN45 cell and do not add positive gram of anti-prostate specific membrane antigen single domain heavy chain antibody phage
Grand cell climbing sheet is negative, shows the anti-prostate specific membrane antigen single domain heavy chain antibody phage positive colony that elutriation goes out
Effectively can express positive cell with PSMA and occur specific binding.
Embodiment 4
The expression in escherichia coli of the single domain heavy chain antibody of anti-prostate specific membrane antigen
In extraction embodiment 2, the phasmid for the single domain heavy chain antibody positive colony of prostate specific membrane antigen is mould
Plate, designs primer amplified genes of interest, amplification condition: 94 DEG C of 5min denaturations；94℃30s、55℃30s、72℃40s
Totally 30 circulations；Last 72 DEG C re-extend 5min.With 1% agarose gel electrophoresis detection after end, and cut glue recovery purpose sheet
Section.
To obtain encoding the double digestion base of the single domain heavy chain antibody of anti-prostate specific membrane antigen provided by the present invention
Because of fragment, clone is connected to expression vector pET-28a, after sequence verification, obtains recombiant plasmid.
Recombinant plasmid transformed is in e. coli bl21 (Rosseta), and picking list bacterium colony carries out abduction delivering.By list bacterium
Fall to being inoculated in the test tube of LB culture medium, 37 DEG C of shaken cultivation, activated overnight；Next day, transfer in fresh LB in the ratio of 1%
In fluid medium, 37 DEG C, 250rpm shaken cultivation, to OD600It is about after 0.6, adds the IPTG of final concentration of 0.1mM, 37
DEG C, 250rpm induce 4h.
The 2mL bacterium solution of above-mentioned induction obtains thalline by 8000rpm is centrifugal, and thalline uses aseptic PBS to wash 3 times, and uses
The aseptic PBS of 1mL carries out resuspended thalline, and ultrasonication thalline is until bacterium solution is limpid on ice, at 4 DEG C, cell pyrolysis liquid is carried out from
The heart, centrifugal condition is 12000rmp/min, and the time is 10min, takes supernatant and adds 5 μ l 5 × SDS sample-loading buffers, and boiling water boils
5min, takes supernatant after being centrifuged and carries out SDS-PAGE electrophoretic analysis and use nickel post to be purified it.
By optimizing abduction delivering condition (such as Host Strains, expression vector, induction time, inducing temperature and IPTG concentration
Deng), the expression of destination protein (single domain heavy chain antibody) can be improved further, resist for preparing anti-prostate specific membrane in a large number
Former single domain heavy chain antibody provides approach.
Embodiment 5
Mensuration for the single domain heavy chain antibody affinity costant of prostate specific membrane antigen
Biotinylation kit is used to carry out biotinylation labelling, thereafter the single domain heavy chain antibody expressed in embodiment 4
The competitive ELISA technical measurement biotinylation of use standard is for the single domain heavy chain antibody of prostate specific membrane antigen and reality
Execute psma protein affinity recombinant expressed in example 1.Concretely comprise the following steps: biotinylated for prostate initially with 1nM
The single domain heavy chain antibody of specific membrane antigen respectively with the PSMA antigen of 13 kinds of variable concentrations (0.1nM～100 μMs) in EP pipe
Carry out 30min to hatch；Thereafter, 90 μ L mixed liquors additions have been used enzyme that 3%BSA-PBST closes, that be coated with psma protein
In target, after hatching 10min, inhale and abandon reactant liquor, and clean with PBST；Then, adding 100 μ L dilution factors is 1: 2000HRP mark
The Streptavidin of note, uses PBST to clean 5 times after hatching 1h；The use of TMB working solution and OD450Mensuration with embodiment 2.
Nonlinear regression analysis is used to obtain OD450During maximum half, corresponding PSMA concentration, according to Ag-Ab competitive binding
The principle of experiment show that the biotinylated single domain heavy chain antibody affinity costant for prostate specific membrane antigen is 5 × 10-7/
About M.
The preparation of the immune affine adsorbing material of embodiment 6
1, the preparation of immune affine magnetic bead
Use nanometer magnetic bead as carrier, after coupling is for the single domain heavy chain antibody of prostate specific membrane antigen, obtain
For the single domain heavy chain antibody immunomagnetic beads of prostate specific membrane antigen, concrete preparation method is as follows:
Take the magnetic bead (transporting nanosecond science and technology company limited, carboxyl magnetic bead 300nm purchased from Wuxi hundred) of 1mg carboxyl modified in centrifugal
Guan Zhong, adds 500 μ l activation buffer (10mM, NaH2PO4, pH 6.0), vortex mixed is uniform, and magnetic frame reclaims magnetic bead, then uses
Activation buffer washs 2 times.It is separately added into 2mg carbodiimide (EDC) and N-hydroxy-succinamide (NHS), after vortex mixed,
Stand 30min.With coupling buffer (10mM, Na2HPO4, pH 7.4) and wash magnetic bead 3 times, add the pin being dissolved in coupling buffer
Single domain heavy chain antibody 1mg, room temperature reaction 3h to prostate specific membrane antigen, washs magnetic bead 3 times with coupling buffer, adds
The 500 μ l coupling buffer containing 1% (w/v) bovine serum albumin (BSA) or 1% (w/v) ovalbumin (OVA) closes unreacted
Active group, room temperature reaction 30min.Magnetic bead is washed 3 times, PBS solution (10mM, pH7.4,0.02%w/ with coupling buffer
V, Na3N) it is stored in 4 DEG C after resuspended.
2, for the single domain heavy chain antibody affine adsorbing material of immunity and the preparation of affinity column of prostate specific membrane antigen.
Using agarose microbeads as carrier, the single domain heavy chain antibody of coupling prostate specific membrane antigen, concrete preparation method is as follows:
The dry glue 0.1M HCl activated by CNBr washs 10 times, balances 5min every time.With coupling buffer (10mM,
Na2HPO4, pH 7.4) wash 10 times, add single domain heavy chain antibody (the 2mg/ every gram agar for prostate specific membrane antigen
Sugar microsphere), room temperature reaction 4h, make the single domain heavy chain antibody for prostate specific membrane antigen coagulate with the agarose of CNBr activation
Glue microsphere covalent coupling.With coupling buffer (10mM, Na2HPO4, pH 7.4) washing 2 times after, add confining liquid room temperature reaction 2h
To close unreacted active group.The phosphate buffer (10mM, pH 7.4) long-pending with 5 times of colloids and acetate buffer solution (0.1M,
PH 4.0) alternately washing 3 times, obtain the covalent coupling immunity parent for the single domain heavy chain antibody of prostate specific membrane antigen
And adsorbing material.Taking the 0.2ml affine adsorbing material of above-mentioned immunity is the chromatographic column of 1ml in capacity, 5～10 times of bed volumes
After PBS (10mM, pH 7.4) washing, add 20% ethanol solution, 4 DEG C of preservations.
3, for the single domain heavy chain antibody affine adsorbing material of immunity and the preparation of affinity column of prostate specific membrane antigen.
Use silica gel microball as carrier, the single domain heavy chain antibody of the anti-prostate specific membrane antigen of coupling, concrete preparation method is as follows:
Take 2g silica gel microball pure water and phosphate buffer (PBS, 10mM, pH 6.0) alternately washing 5～10 times, use 10ml
PBS suspension silica gel microball, adds the 5mg single domain heavy chain antibody for prostate specific membrane antigen, mixing, adds eventually
The carbodiimide (EDC) of concentration 5mg/ml, mixes rapidly, 4 DEG C of stirring reactions 12～24h, obtains covalent coupling for prostatitis
The affine adsorbing material of immunity of the single domain heavy chain antibody of gland specific membrane antigen.Take the 0.2ml affine adsorbing material of above-mentioned immunity in
Capacity is the chromatographic column of 1ml, after PBS (10mM, the pH 6) washing of 5～10 times of bed volumes, adds containing 0.02% (w/v) Na3N
PBS (10mM, pH 6), 4 DEG C of preservations.
The immunity affine adsorbing material aglucon that the present invention is directed to prostate specific membrane antigen is single domain heavy chain antibody, has
Aminoacid sequence shown in SEQ ID NO.:1, this aglucon can specific recognition prostate specific membrane antigen.This single domain heavy chain
Antibody is readily available, adsorption efficiency high, and can produce aglucon by biological method mass propgation is single domain heavy chain antibody, it is to avoid
The loaded down with trivial details production methods such as artificial antibody, greatly reduce production cost.There is acid and alkali-resistance, high temperature resistant and the spy such as be readily produced
Property, these characteristics have for the low cost of prostate specific membrane antigen, reusable purification and immunological detection method
Important practical value.
Claims (9)
1., for the single domain heavy chain antibody of prostate specific membrane antigen, there is the aminoacid sequence shown in SEQ ID NO.:1.
2. a nucleic acid molecules, is characterized in that encoding aminoacid sequence described in claim 1.
Nucleic acid molecules the most according to claim 2, it is characterised in that sequence such as SEQ ID NO.:2.
4. the carrier of the nucleotide sequence comprised described in claim 2.
5. the host cell of the carrier comprised described in claim 4.
6. described in claim 1 for prostate specific membrane antigen single domain heavy chain antibody at immune detection, thalline or antigen
Application in enriching and purifying.
7. the single domain heavy chain antibody for prostate specific membrane antigen described in claim 1 is preparing prostate specific membrane
Application in antigen immune detection, enrichment and purified reagent or material.
8. the single domain heavy chain antibody for prostate specific membrane antigen described in claim 1 is by random or rite-directed mutagenesis skill
Art carry out house of correction acquisition can be specific binding with prostate specific membrane antigen antibody.
9., for the affine adsorbing material of immunity of prostate specific membrane antigen, including carrier, be mounted on carrier joins
Base, it is characterised in that this material is using the single domain heavy chain antibody for prostate specific membrane antigen as aglucon, described for front
The single domain heavy chain antibody of row gland specific membrane antigen has the aminoacid sequence shown in SEQ ID NO.:1.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2020530285A (en) * | 2017-07-10 | 2020-10-22 | クレッシェンド、バイオロジックス、リミテッドＣｒｅｓｃｅｎｄｏ Ｂｉｏｌｏｇｉｃｓ Ｌｉｍｉｔｅｄ | Therapeutic PSMA binding molecule |
| US11746158B2 (en) | 2016-01-12 | 2023-09-05 | Crescendo Biologics Limited | Therapeutic molecules |
| US11866510B2 (en) | 2016-05-06 | 2024-01-09 | Crescendo Biologics Limited | Chimeric antigen receptor with single domain antibody |
| US12077595B2 (en) | 2017-11-13 | 2024-09-03 | Crescendo Biologics Limited | Single domain antibodies that bind to CD137 |
-
2016
- 2016-02-03 CN CN201610079410.9A patent/CN105968205B/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11746158B2 (en) | 2016-01-12 | 2023-09-05 | Crescendo Biologics Limited | Therapeutic molecules |
| US11866510B2 (en) | 2016-05-06 | 2024-01-09 | Crescendo Biologics Limited | Chimeric antigen receptor with single domain antibody |
| JP2020530285A (en) * | 2017-07-10 | 2020-10-22 | クレッシェンド、バイオロジックス、リミテッドＣｒｅｓｃｅｎｄｏ Ｂｉｏｌｏｇｉｃｓ Ｌｉｍｉｔｅｄ | Therapeutic PSMA binding molecule |
| JP7346378B2 (en) | 2017-07-10 | 2023-09-19 | クレッシェンド、バイオロジックス、リミテッド | Therapeutic PSMA binding molecules |
| US12077595B2 (en) | 2017-11-13 | 2024-09-03 | Crescendo Biologics Limited | Single domain antibodies that bind to CD137 |
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