CN105622755B - A kind of nano antibody of anti-prostate-specific membrane antigen extracellular region - Google Patents

A kind of nano antibody of anti-prostate-specific membrane antigen extracellular region Download PDF

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CN105622755B
CN105622755B CN201610074011.3A CN201610074011A CN105622755B CN 105622755 B CN105622755 B CN 105622755B CN 201610074011 A CN201610074011 A CN 201610074011A CN 105622755 B CN105622755 B CN 105622755B
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prostate
specific membrane
membrane antigen
heavy chain
single domain
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范校周
郭燕丽
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First Affiliated Hospital of TMMU
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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

A kind of nano antibody of anti-prostate-specific membrane antigen extracellular region
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 extracellular region), there is amino acid sequence shown in SEQ ID NO.:1, amino acid sequence Stroke with structural domain can be numbered by standardized antibody amino acids sequence method for numbering serial (ImMunoGeneTics, IMGT) Point.
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 Res 38 (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):
ATGGCCCAGGTGCAGCTCGTGGAGTCGGGGGGAGGCTTGGTGCAGGCTGGGGATTCTCTGAGACTCTCC TGTGCAGCCTCTAGAAGGACGTTCAGTTTCATGGCATGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGC AGCTATTACTAGTGGTGGTCGTAATACAAACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATG CCAAGAACACGATGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTAACGCGGCGGCC CGGTGGAATAACTACTGGGGCCAGGGGACCCCGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAGCGGC CGC
The amino acid sequence that it is encoded is as shown in SEQ ID NO.:1:
QVQLVESGGGLVQAGDSLRLSCAASRRTFSFMAWYRQAPGKQRELVAAITSGGRNTNYADSVKGRFTI SRDNAKNTMYLQMNSLKPEDTAVYYCNAAARWNNYWGQGTPVTVSS.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.

Claims (6)

1. being directed to the single domain heavy chain antibody of prostate-specific membrane antigen, amino acid sequence is as shown in SEQ ID NO .:2.
2. a kind of nucleic acid molecules, it is characterized in that amino acid sequence described in coding claim 1.
3. a kind of carrier comprising nucleic acid molecules as claimed in claim 2.
4. a kind of host cell comprising carrier as claimed in claim 3.
5. the single domain heavy chain antibody described in claim 1 for prostate-specific membrane antigen is preparing prostate specific membrane Application in antigen immune detection, enrichment and purified reagent or material.
6. a kind of affine in immunity adsorbent material for prostate-specific membrane antigen, including carrier, and be mounted on carrier Aglucon, it is characterised in that the material is made with the single domain heavy chain antibody described in claim 1 for prostate-specific membrane antigen For aglucon.
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