CN105968205B - A Nanobody Against Prostate Specific Membrane Antigen - Google Patents
A Nanobody Against Prostate Specific Membrane Antigen Download PDFInfo
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- CN105968205B CN105968205B CN201610079410.9A CN201610079410A CN105968205B CN 105968205 B CN105968205 B CN 105968205B CN 201610079410 A CN201610079410 A CN 201610079410A CN 105968205 B CN105968205 B CN 105968205B
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- specific membrane
- membrane antigen
- prostate
- heavy chain
- chain antibody
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- 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
- C07K16/3069—Reproductive system, e.g. ovaria, uterus, testes, prostate
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2220/00—Aspects relating to sorbent materials
- B01J2220/40—Aspects relating to the composition of sorbent or filter aid materials
- B01J2220/48—Sorbents characterised by the starting material used for their preparation
- B01J2220/4812—Sorbents characterised by the starting material used for their preparation the starting material being of organic character
- B01J2220/4856—Proteins, DNA
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
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Abstract
The invention belongs to genetic engineering fields, are specially directed to the single domain heavy chain antibody of prostate-specific membrane antigen, with amino acid sequence shown in SEQ ID NO.1, can be used for the fields such as immune detection, antigen enrichment purifying.Amino acid sequence provided by the present invention can be used as precursor, it is transformed by random or site-directed mutagenesis technique, property (compatibility, specificity, stability etc.) better mutant can be obtained, is further used for medicine, industry, agriculture protein or polypeptide for developing.
Description
Technical field
The present invention relates to single domain heavy chain antibody technology (also known as nano antibody technology) and genetic engineering antibody technologies, special
It is not the single domain heavy chain antibody or polypeptide for prostate-specific membrane antigen.
Technical background
Single domain antibody refers to the genetic engineering antibody being made of common antibody variable region (VH or VL).Single domain heavy chain antibody
(also known as nano antibody, VHH antibody, variable domain of heavy chain of heavy-chain
Antibody) refer to and be only made of the variable region heavy chain antibody (heavy-chain antibody) (Variable region)
Genetic engineering antibody, wherein heavy chain antibody (heavy-chain antibody) is that one kind is present in the animal bodies such as camel, shark
The antibody of interior natural deletions light chain.Single domain heavy chain antibody is the smallest intact antigen binding fragment being currently known, and has molecule
The features such as measuring small, good penetrability is widely used in the fields such as basic research, medical diagnosis and detection, antibody drug exploitation at present.
Prostate cancer is a kind of malignant tumour for seriously threatening elderly men health, and early diagnosis and therapy is pre- for its
After have great importance.Prostate-specific membrane antigen (prostate specific membrane antigen, PSMA)
It is a kind of II type transmembrane glycoprotein on prostatic cell film, is made of 750 amino acid, is divided into intracellular region (amino acid
Sequence is 1-18), transmembrane region (19-43) and extracellular region (44-750), relative to the prostate-specific for being conventionally used to clinical detection
Antigen (prostate specific antigen, PSA), is a kind of more sensitive and special prostate cancer marker,
It is high expression especially in hormone-refractory prostate cancer and prostate cancer transfer stove, is distinguishing prostate cancer and other classes
The susceptibility and specificity of type malignant tumour are respectively 65.9% and 94.5%.In addition, the entity in a variety of non-prostate sources
Tumor, such as lung cancer, bladder cancer, gastric cancer, cancer of pancreas, kidney and colorectal cancer, PSMA are also expressed in tumor vessel to high special
On endothelial cell.And itself there are the activity of neural carboxypeptidase and folic acid hydrolase, the born of the same parents that 707 amino acid forms in addition
Outskirt is capable of providing the features such as multiple epitopes, has become important in tumour immunity targeted therapy and molecular imaging
Study target spot.
At present, it has been found that it is a variety of to be also prepared for a variety of substances that specifically bound with it for PSMA, it wraps
Monoclonal antibody 7E11, J591, aptamer A9 and A10 are included, ScFv antibody D7, Fab antibody and the humanization that recombinant technique obtains are anti-
The report of body is especially sprayed by U.S. FDA approval for the diagnosis of prostate cancer and the 111In- of advanced stage radionuclide therapy
Ground peptide, the monoclonal antibody being namely based on for PSMA are connected with radionuclide.But compared with single domain heavy chain antibody, this
The disadvantages of that there are production costs is relatively high for a little ligands, and preparation process is complicated.
Summary of the invention
The object of the present invention is to provide the single domain heavy chain antibodies for being directed to prostate-specific membrane antigen, can be used to prepare
Detect the reagent and tool of prostate-specific membrane antigen.
The present invention provides a kind of single domain heavy chain antibody (the anti-forefront i.e. of the invention for being directed to prostate-specific membrane antigen
The nano antibody of gland specific membrane antigen), there is amino acid sequence shown in SEQ ID NO.:1, amino acid sequence can pass through
The division with structural domain is numbered in standardized antibody amino acids sequence method for numbering serial (ImMunoGeneTics, IMGT).
The present invention provides a kind of protein or polypeptide, it is characterized in that including ammonia more than one or two in framework region
Base acid sequence, and at least there is 90% homology with an amino acid sequence.
The present invention provides a kind of protein or polypeptide, it is characterized in that comprising more than one or two in complementary determining region
Amino acid sequence, and at least with an amino acid sequence have 80% homology.
The present invention provides a nucleic acid molecules, it is characterized in that coding SEQ ID NO.:1, it can be with by genetic codon
When obtain the particular sequences of the nucleic acid molecules.The sequence of the nucleic acid molecules such as SEQ ID NO.:2.
The present invention also provides a nucleic acid molecules, it is characterized in that coding SEQ ID NO.:1 partial domain, passes through heredity
Codon can obtain the particular sequence of the 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
Up to obtain corresponding protein or polypeptide.These expression systems include bacterium, saccharomycete, filamentous fungi, zooblast, insect
Cell, plant cell or Cell free expression system.
The present invention also provides a kind of carriers, include the nucleic acid sequence.Since genetic codon has degeneracy, the nucleic acid
Sequence can be different according to different application purposes.
The present invention also provides a kind of host cells, including the protein or expression vector.
The present invention also provides a kind of methods of prostate-specific membrane antigen on detection cell, it is characterized in that containing above-mentioned egg
White matter or polypeptide.Ability based on protein provided by the invention or polypeptide and prostate-specific membrane antigen specific binding,
Establish 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 for prostate-specific membrane antigen single domain heavy chain antibody non-disease diagnoses and treatment purpose immune detection,
And the application in thallus or antigen enrichment purifying.
Amino acid sequence provided by the present invention can be used as precursor, is transformed by random or site-directed mutagenesis technique,
Property (water solubility, stability, affinity and specificity etc.) better mutant can be obtained, is further used for for developing
Medicine, industry, agriculture protein or polypeptide.
The invention further relates to a kind of affine in immunity adsorbent materials for prostate-specific membrane antigen, including carrier, take
The aglucon being loaded on carrier, the material is using the single domain heavy chain antibody for prostate-specific membrane antigen as aglucon, the needle
There is amino acid sequence shown in SEQ ID NO.:1 to the single domain heavy chain antibody of prostate-specific membrane antigen.The carrier is
Magnetic bead, agarose gel microsphere, silica gel microball or porous material.
Some terms that the present invention is described have following meaning:
Homology: the similarity degree of two or more amino acid sequences, first amino acid sequence and second ammonia are described
The percentage of homology can be by [in first amino acid sequence corresponding to second amino acid sequence between base acid sequence
The quantity of the identical amino acid residue of amino acid sequence at position] exist divided by [amino acid sum in first amino acid sequence]
Calculated multiplied by [100%], wherein the missing of some amino acid in second amino acid sequence, insertion, replacement or addition (with
First amino acid is compared) it is considered as having difference.Alternatively, percent homology also can use known for sequence
The Computing program matched such as NCBI Blast is obtained.
Structural domain: the fundamental structural unit of tertiary protein structure usually has the function of certain.
IMGT number: one of IMGT database (The International ImMunoGeneTics Datbase)
Normalised antibody amino acids sequence method for numbering serial.Specific method for numbering serial can with bibliography (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
27 (1): the description in 55-77.http: //www.imgt.org/).
Codon (codon): also known as three disjunctor codons (triplet code), refer to the core corresponding to certain amino acid
Thuja acid triplet.The position of polypeptide chain in this kind of amino acid insertion growth is determined during translation.
Beneficial effects of the present invention: the present invention has for the single domain heavy chain antibody or polypeptide of prostate-specific membrane antigen
It is specifically bound with prostate-specific membrane antigen, biological method large-scale production, at low cost, efficient, molecule can be passed through
The properties such as small, good penetrability are measured, show good application prospect.
Detailed description of the invention
Fig. 1 colony PCR product electrophoresis, recombinant protein electrophoresis and Western Blot qualification figure.Left side Marker swimming lane is
DNA molecular amount standard, the bacterium colony PCR fragment in swimming lane 1 appear in desired location.Centre carries out SDS- for albumen after purification
There is bright band in desired location in PAGE detection.The right is with anti-PSMA monoclonal antibody and anti-His tag antibody
Western Blot qualification figure.
Specific embodiment
Below by the preparation, analysis and application of single domain heavy chain antibody (polypeptide), the present invention will be further described, these
Specific embodiment is not construed in any way as limiting application range of the invention.
Embodiment 1
The eukaryotic expression of prostate-specific membrane antigen film outskirt
Total serum IgE in the LNCaP cell of high expression PSMA is extracted using RNAiso Plus reagent, is obtained using RT-PCR method
To the DNA fragmentation of coding PSMA extracellular domain fragment, eukaryon table is inserted into using Not I and two kinds of restriction enzymes of BamH I
Up to carrier pRAG2a, recombinant plasmid is connected as by T4DNA ligase.Recombinant plasmid thermal shock is transformed into TOP10 competent cell
Overnight incubation, by identification, correctly clone send sequence verification.The plasmid for extracting positive colony, uses liposome
DNA plasmid is transfected into HEK-293 cell and is cultivated by Lipofectamine 2000, is collected supernatant in different time phases, is carried out
SDS-PAGE electrophoresis detection.Culture after a certain period of time, operates according to HisTrap FF Crude kit and purifies the albumen.It will be pure
After albumen after change carries out SDS-PAGE, electricity is gone on pvdf membrane, after the closing of 5% skimmed milk power, be separately added into PSMA antibody and
His antibody, 4 DEG C overnight；After rinsing, add secondary antibody to be incubated for 1h at room temperature, rinse again, developing solution is added and is developed (Fig. 1).
Embodiment 2
Anti- prostate-specific membrane antigen single domain heavy chain antibody is (i.e. anti-for anti-prostate-specific membrane antigen single domain heavy chain
Body) elutriation and identification
It uses the method for solid phase elutriation to apply from hunchbacked source natural antibody phage display library (for bibliography: " to chase after, Xu Yang,
Liu Xia waits building and identification [J] Chinese biological engineering magazine in camel source natural single domain heavy chain antibody library, 2011,31 (4): 31-
36. " in construct display libraries) in elutriation be directed to prostate-specific membrane antigen single domain heavy chain antibody.Prostate specific
The expression of membranous antigen film outskirt is carried out according to the above embodiments 1, when first round elutriation, using phosphate buffer solution (PBS,
PH7.4) to 150 μ g/mL, (it is respectively 100,50,50 μ that 2-4 takes turns peridium concentration to the PSMA extracellular region protein of the above-mentioned synthesis of dilution
G/mL), 100 μ L are added in every hole on ELISA Plate, and 4 DEG C of coatings are overnight.PBST (containing 0.5%Tween 20) board-washing 5 times, 3%
Board-washing 3 times, the 100 μ L of phage antibody library being incubated for 0.5%BSA-PBS is added (containing about 2 in 37 DEG C of closing 2h in BSA-PBS
×1011CFU), 37 DEG C of incubation 1h, with PBST board-washing 5 times (increasing by 3 times by wheel), then with PBS board-washing 10 times (increasing by 5 times by wheel).
Again with the bacteriophage (37 DEG C, 5min) of 100 μ L eluents (glycine-HCI, pH 2.2) elution 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 after the amplification of remaining eluate for next round
Choosing.
Use concentration for the PSMA extracellular region protein coated elisa plate of 5 μ g/mL after four-wheel elutriation, board-washing, closing are same
On.37 DEG C of incubation 15min of phage clone of amplification purification, 100 holes μ L/, 37 DEG C of incubation 1h are added.Dilution is added after board-washing
For 1: 5000 HRP-anti-M13 antibody, 100 holes μ L/, 37 DEG C of incubation 1h.PBST board-washing 5 times, add 100 μ L/ of TMB working solution
Hole, room temperature 20min, every hole are added 50 μ L sulfuric acid (concentration 2mol/L) and terminate reaction, measure 450nm light absorption value.With previous round
The phage antibody library direct coated ELISA Plate of amplification is used as positive control using PBS substitution phage clone as blank control
The combination activity of indirect phage-ELISA method measurement phage particle.
The indirect phage-ELISA of table 1 is loaded table
It send sequencing company to carry out sequencing ELISA positive colony, obtains anti-prostate-specific membrane antigen single domain weight
The Insert Fragment DNA sequence dna of chain antibody bacteriophage positive colony, specially (SEQ ID NO.:2):
ATGGCCCAGGTGCAGCTGGTGGAGTCGGGGGGGGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCC
TGTGCAGCCTCTGGACGCACCAAAAGTACCAGTTCTATGGGCTGGTTCCGCCAGGCTCCAGGAAAGGGGCGTGACTT
TGTAGCAGGTATTAGTGGTGCAGGTAAAACAAACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACG
GCGCCAAGAACACGGTGCATCTGCAAATGAACAGCATGAAACCTGAGGACACGGCCGTCTATTACTGTAACACCCTG
ATTCGACCCCGGGGGTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAGCGGC
CGC
The amino acid sequence that it is encoded is as shown in SEQ ID NO.:1:
QVQLVESGGGLVQAGGSLRLSCAASGRTKSTSSMGWFRQAPGKGRDFVAGISGAGKTNYADSVKGRFT
ISRDGAKNTVHLQMNSMKPEDTAVYYCNTLIRPRGWGQGTQVTVSS.Anti- prostate-specific membrane antigen i.e. of the invention
Single domain heavy chain antibody can be specifically bound with the film outskirt of psma protein.
Embodiment 3
The ELISA and fluorescence immunoassay of PSMA expression cell are detected
Gastric cancer cell MKN45 does not express PSMA, and prostate gland cancer cell LNCaP expresses PSMA, by taking both cells as an example,
Cell is carried out using the anti-prostate-specific membrane antigen single domain heavy chain antibody bacteriophage positive colony that elutriation in embodiment 2 obtains
Horizontal ELISA and fluorescence immunoassay detection.It is planted respectively into 96 orifice plates into LNCaP cell (expression PSMA) and MKN45 cell is (no
Express PSMA) each 1 × 104It is a, overnight incubation.4% paraformaldehyde is fixed, and the 3% hydrogen peroxide liquid of 100 μ L is added dropwise in every hole, is used
To block endogenous peroxidase activity, 37 DEG C of incubation 30min.TBS board-washing 3 times, 100 μ L are added in 5%BSA-PBS closing
Anti- prostate-specific membrane antigen single domain heavy chain antibody bacteriophage positive colony, in 37 DEG C of incubation 1h.Thereafter PBS is rinsed, addition
HRP-anti-M13 antibody, TMB working solution and OD450Measurement with embodiment 2.Positive control substitutes cell using bacteriophage, empty
White control is repeated 3 times using the phage clone of PBS substitution addition.
It plants after adding cell climbing sheet in every hole into 24 orifice plates into LNCaP cell and MKN45 cell each 4 × 105It is a, training
It supports overnight.Creep plate is taken out, 4% paraformaldehyde is fixed, and is rinsed, and 3% hydrogen peroxide is added dropwise on creep plate surface, to block endogenous
Peroxidase activity.Thereafter TBS is washed 3 times, and 5%BSA-PBS closes 30min.100 μ L are added dropwise and show that nanometer of the present invention is anti-
The phage clone of body is incubated for 1h, the cell climbing sheet of phage clone is not added as blank control.Thereafter addition dilution is 1:
2000 anti-M13 monoclonal antibody is incubated for 30min.Be added dropwise after developing a film 30 μ l dilutions be 1: 200 FITC- goat resist it is small
Mouse secondary antibody is protected from light and is incubated for 30min, and redyed with DAPI dyeing liquor, is placed in fluorescence microscopy under the microscope.
The result shows that: the MKN45 cell measured value due to not expressing PSMA statistically has differences with blank control, table
With it non-specific binding can occur for the bright bacteriophage；But LNCaP cell measured value is significantly higher than MKN45 cell, shows this
Specificity of a part of difference from PSMA and anti-prostate-specific membrane antigen single domain heavy chain antibody bacteriophage 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 not plus positive gram of anti-prostate-specific membrane antigen single domain heavy chain antibody bacteriophage
Grand cell climbing sheet is feminine gender, shows the anti-prostate-specific membrane antigen single domain heavy chain antibody bacteriophage positive colony that elutriation goes out
Positive cell effectively can be expressed with PSMA to specifically bind.
Embodiment 4
Expression of the single domain heavy chain antibody of anti-prostate-specific membrane antigen in Escherichia coli
Extracting the phasmid in embodiment 2 for the single domain heavy chain antibody positive colony of prostate-specific membrane antigen is mould
Plate designs primer amplified target gene, amplification condition: 94 DEG C of 5min initial denaturations；94℃30s,55℃30s,72℃40s
Totally 30 circulations；Last 72 DEG C re-extend 5min.After detected with 1% agarose gel electrophoresis, and gel extraction purpose piece
Section.
The double digestion base of the single domain heavy chain antibody of anti-prostate-specific membrane antigen provided by the present invention will be obtained encoding
Because of segment, clone is connected to expression vector pET-28a, after sequence verification, obtains recombinant plasmid.
Recombinant plasmid transformed is in e. coli bl21 (Rosseta), and picking single colonie carries out inducing expression.By single bacterium
It falls in the test tube for being inoculated in LB culture medium, 37 DEG C of shaken cultivations are activated overnight；Next day transfers in 1% ratio in fresh LB
In fluid nutrient medium, 37 DEG C, 250rpm shaken cultivation, until OD600After about 0.6, it is added the IPTG of final concentration of 0.1mM, 37
DEG C, 250rpm induce 4h.
The 2mL bacterium solution of above-mentioned induction is centrifuged to obtain thallus by 8000rpm, and thallus is washed 3 times using sterile PBS, is used in combination
The sterile PBS of 1mL carries out resuspension thallus, on ice ultrasonication thallus until bacterium solution it is limpid, at 4 DEG C to cell pyrolysis liquid carry out from
The heart, centrifugal condition 12000rmp/min, time 10min take supernatant that 5 μ l 5 × SDS sample-loading buffers are added, and boiling water boils
5min takes supernatant to carry out SDS-PAGE electrophoretic analysis and purify using nickel column to it after centrifugation.
By optimizing inducing expression condition (such as host strain, expression vector, induction time, inducing temperature and IPTG concentration
Deng), it can be further improved the expression quantity of destination protein (single domain heavy chain antibody), it is anti-largely to prepare anti-prostate specific membrane
Former single domain heavy chain antibody provides approach.
Embodiment 5
For the measurement of the single domain heavy chain antibody affinity costant of prostate-specific membrane antigen
The single domain heavy chain antibody expressed in embodiment 4 is subjected to biotinylation label using biotinylation kit, thereafter
The single domain heavy chain antibody and reality of prostate-specific membrane antigen are directed to using the competitive ELISA technology measurement biotinylation of standard
Apply the psma protein affinity recombinantly expressed in example 1.Specific steps are as follows: be directed to prostate using 1nM is biotinylated first
The single domain heavy chain antibody of specific membrane antigen respectively the PSMA antigen with 13 kinds of various concentrations (0.1nM~100 μM) in EP pipe
Carry out 30min incubation；Thereafter, enzyme that is closed using 3%BSA-PBST, being coated with psma protein is added in 90 μ L mixed liquors
In target, after being incubated for 10min, inhales and abandon reaction solution, and cleaned with PBST；Then, it is 1: 2000HRP mark that 100 μ L dilutions, which are added,
The Streptavidin of note is cleaned 5 times after being incubated for 1h using PBST；The use of TMB working solution and OD450Measurement with embodiment 2.
OD is obtained using nonlinear regression analysis450When maximum value 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/
M or so.
The preparation of 6 affine in immunity adsorbent material of embodiment
1, the preparation of affine in immunity magnetic bead
It is obtained after coupling is for the single domain heavy chain antibody of prostate-specific membrane antigen using nanometer magnetic bead as carrier
For the single domain heavy chain antibody immunomagnetic beads of prostate-specific membrane antigen, it is specific the preparation method is as follows:
Take the magnetic bead (transporting nanosecond science and technology Co., Ltd, carboxyl magnetic bead 300nm purchased from Wuxi hundred) of 1mg carboxyl modified in centrifugation
500 μ l activation buffer (10mM, NaH are added in Guan Zhong2PO4, pH 6.0), vortex mixed is uniform, and magnetic frame recycles magnetic bead, then uses
Activation buffer washs 2 times.It is separately added into 2mg carbodiimide (EDC) and n-hydroxysuccinimide (NHS), after vortex mixed,
Stand 30min.With coupling buffer (10mM, Na2HPO4, pH 7.4) and washing magnetic bead 3 times, the needle for being dissolved in coupling buffer is added
To the single domain heavy chain antibody 1mg of prostate-specific membrane antigen, 3h is reacted at room temperature, is washed magnetic bead 3 times with coupling buffer, is added
Coupling buffer of the 500 μ l containing 1% (w/v) bovine serum albumin(BSA) (BSA) or 1% (w/v) ovalbumin (OVA) closes unreacted
Active group, react at room temperature 30min.Magnetic bead 3 times are washed with coupling buffer, PBS solution (10mM, pH7.4,0.02%w/
V, Na3N 4 DEG C are stored in after) being resuspended.
2, for the single domain heavy chain antibody affine in immunity adsorbent material of prostate-specific membrane antigen and the preparation of affinity column.
Using agarose microbeads as carrier, be coupled the single domain heavy chain antibody of prostate-specific membrane antigen, it is specific the preparation method is as follows:
The CNBr dry glue activated is washed 10 times with 0.1M HCl, balances 5min every time.With coupling buffer (10mM,
Na2HPO4, pH 7.4) and washing 10 times, single domain heavy chain antibody (the every gram of agar of 2mg/ for being directed to prostate-specific membrane antigen is added
Sugared microballoon), 4h is reacted at room temperature, keeps the agarose of single domain heavy chain antibody and the CNBr activation for prostate-specific membrane antigen solidifying
Glue microballoon covalent coupling.With coupling buffer (10mM, Na2HPO4, pH 7.4) washing 2 times after, be added confining liquid react at room temperature 2h
To close unreacted active group.With 5 times of colloids product phosphate buffer (10mM, pH 7.4) and acetate buffer solution (0.1M,
PH 4.0) alternately washing 3 times, covalent coupling is obtained for the immune parent of the single domain heavy chain antibody of prostate-specific membrane antigen
And adsorbent material.Take the above-mentioned affine in immunity adsorbent material of 0.2ml in the chromatographic column that capacity is 1ml, 5~10 times of bed volumes
After PBS (10mM, pH 7.4) washing, 20% ethanol solution, 4 DEG C of preservations are added.
3, for the single domain heavy chain antibody affine in immunity adsorbent material of prostate-specific membrane antigen and the preparation of affinity column.
Using silica gel microball as carrier, be coupled the single domain heavy chain antibody of anti-prostate-specific membrane antigen, it is specific the preparation method is as follows:
Alternately washing 5~10 times of 2g silica gel microball pure water and phosphate buffer (PBS, 10mM, pH 6.0) are taken, 10ml is used
The single domain heavy chain antibody that 5mg is directed to prostate-specific membrane antigen is added in PBS buffer solution suspension silica gel microball, mixes, is added eventually
The carbodiimide (EDC) of concentration 5mg/ml mixes rapidly, 4 DEG C be stirred to react 12~for 24 hours, obtain covalent coupling for forefront
The affine in immunity adsorbent material of the single domain heavy chain antibody of gland specific membrane antigen.Take the above-mentioned affine in immunity adsorbent material of 0.2ml in
Capacity is that the chromatographic column of 1ml is added after PBS (10mM, the pH 6) washing of 5~10 times of bed volumes and contains 0.02% (w/v) Na3N
PBS (10mM, pH 6), 4 DEG C preservation.
The present invention is single domain heavy chain antibody for the affine in immunity adsorbent material aglucon of prostate-specific membrane antigen, is had
Amino acid sequence shown in SEQ ID NO.:1, the aglucon can specific recognition prostate-specific membrane antigen.The single domain heavy chain
Antibody is easy to get, adsorption efficiency is high, and can produce aglucon by biological method mass propgation is single domain heavy chain antibody, is avoided
The cumbersome production methods such as artificial antibody, greatly reduce production cost.With acid and alkali-resistance, high temperature resistant and the spies such as it is readily produced
Property, these characteristics have inexpensive, the reusable purifying of prostate-specific membrane antigen and immunological detection method
Important practical value.
Sequence table
<110>No.3 Hospital Attached to No.3 Military Medical Univ., P.L.A.
<120>a kind of nano antibody of anti-prostate-specific membrane antigen
<130> 2015
<140> 2016100794109
<141> 2016-02-03
<160> 2
<170> SIPOSequenceListing 1.0
<210> 3
<211> 114
<212> PRT
<213>artificial sequence (nothing)
<400> 3
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Lys Ser Thr Ser
20 25 30
Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Asp Phe Val
35 40 45
Ala Gly Ile Ser Gly Ala Gly Lys Thr Asn Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Gly Ala Lys Asn Thr Val His Leu
65 70 75 80
Gln Met Asn Ser Met Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn
85 90 95
Thr Leu Ile Arg Pro Arg Gly Trp Gly Gln Gly Thr Gln Val Thr Val
100 105 110
Ser Ser
<210> 4
<211> 380
<212> DNA
<213>artificial sequence (nothing)
<400> 4
atggcccagg tgcagctggt ggagtcgggg gggggcttgg tgcaggctgg ggggtctctg 60
agactctcct gtgcagcctc tggacgcacc aaaagtacca gttctatggg ctggttccgc 120
caggctccag gaaaggggcg tgactttgta gcaggtatta gtggtgcagg taaaacaaac 180
tatgcagact ccgtgaaggg ccgattcacc atctccagag acggcgccaa gaacacggtg 240
catctgcaaa tgaacagcat gaaacctgag gacacggccg tctattactg taacaccctg 300
attcgacccc gggggtgggg ccaggggacc caggtcaccg tctcctcaga acccaagaca 360
ccaaaaccac aagcggccgc 380
Claims (7)
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| US10975161B2 (en) | 2016-01-12 | 2021-04-13 | Crescendo Biologics Limited | Molecules that bind prostate specific membrane antigen (PSMA) |
| GB201607968D0 (en) | 2016-05-06 | 2016-06-22 | Crescendo Biolog Ltd | Chimeric antigen receptor |
| GB201711068D0 (en) * | 2017-07-10 | 2017-08-23 | Crescendo Biologics Ltd | Therapeutic molecules binding PSMA |
| EP3710477A1 (en) | 2017-11-13 | 2020-09-23 | Crescendo Biologics Limited | Single domain antibodies that bind to cd137 |
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Non-Patent Citations (2)
| Title |
|---|
| Camel Heavy Chain Antibodies Against Prostate-Specific Membrane Antigen;Mehdi Evazalipour等;《Hybridoma》;20121217;第31卷(第6期);全文 * |
| 前列腺特异性膜抗原胞外区的真核表达及其纳米抗体的淘选;范校周等;《第三军医大学学报》;20131115;第35卷(第21期);全文 * |
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