CN105542008A - Single domain heavy-chain antibody capable of being specifically bound with prostate specific membrane antigen - Google Patents

Single domain heavy-chain antibody capable of being specifically bound with prostate specific membrane antigen Download PDF

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CN105542008A
CN105542008A CN201610073980.7A CN201610073980A CN105542008A CN 105542008 A CN105542008 A CN 105542008A CN 201610073980 A CN201610073980 A CN 201610073980A CN 105542008 A CN105542008 A CN 105542008A
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prostate specific
membrane antigen
specific membrane
single domain
chain antibody
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涂追
许杨
付金衡
陶勇
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Nanchang University
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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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    • G01N33/57407—Specifically defined cancers
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Abstract

The invention belongs to the field of genetic engineering and in particular provides a single domain heavy-chain antibody aiming at a prostate specific membrane antigen. The single domain heavy-chain antibody has an amino acid sequence shown in SEQ ID NO.:1 and can be used in the fields of immunoassay, antigen enrichment and purification, and the like. A mutant with better properties (affinity, specificity, stability, and the like) can be obtained by using the amino acid sequence provided by the invention as a precursor and transforming the amino acid sequence through random or site-directed mutagenesis technology and is used for developing proteins or polypeptides further used in medicines, industry and agriculture.

Description

The single domain heavy chain antibody of energy specific combination prostate specific membrane antigen
Technical field
The present invention relates to single domain heavy chain antibody technology (also known as nano antibody technology), and genetic engineering antibody technology, particularly for single domain heavy chain antibody or the polypeptide of prostate specific membrane antigen.
Technical background
Single domain antibody refers to the genetic engineering antibody be made up of common antibody variable region (VH or VL).Single domain heavy chain antibody (is also called nano antibody, VHH antibody, variabledomainofheavychainofheavy-chainantibody) genetic engineering antibody be only made up of heavy chain antibody (heavy-chainantibody) variable region (Variableregion) is referred to, wherein, heavy chain antibody (heavy-chainantibody) is a kind of antibody being present in natural deletions light chain in the animal body such as camel, shark.Single domain heavy chain antibody is minimum intact antigen binding fragment known at present, has the features such as molecular weight is little, good penetrability, has been widely used in fundamental research, medical diagnosis and the field such as detection, antibody drug exploitation at present.
Prostate cancer is a kind of malignant tumour of serious threat elderly men health, and its early diagnosis and therapy has great importance for its prognosis.Prostate specific membrane antigen (prostatespecificmembraneantigen, PSMA) be a kind of II type transmembrane glycoprotein be positioned on prostatic cell film, be made up of 750 amino acid, be divided into intracellular region (aminoacid sequence is 1-18), cross-film district (19-43) and extracellular region (44-750), relative to the prostate specific antigen (prostatespecificantigen being conventionally used to clinical detection, PSA), it is a kind of more responsive and special prostate cancer marker, especially in hormone-refractory prostate cancer and prostate cancer metastasis, high expression level is, 65.9% and 94.5% is respectively at the susceptibility and specificity of distinguishing prostate cancer and other types malignant tumour.In addition, at the solid tumor in multiple non-prostate gland source, as lung cancer, bladder cancer, cancer of the stomach, carcinoma of the pancreas, kidney and colorectal cancer etc., PSMA is also expressed on tumor vascular endothelial cell high special.And itself there is the activity of neural carboxypeptidase and folic acid lytic enzyme, the extracellular region of 707 amino acid compositions can provide the features such as multiple epitopes in addition, has become research target spot important in tumour immunity targeted therapy and molecular imaging.
At present, it is found that multiple for PSMA also prepare multiple can with the material of its generation specific binding, comprise monoclonal antibody 7E11, J591, fit A9 and A10, the report of ScFv antibody D7, Fab antibody and humanized antibody that recombinant technology obtains, particularly by U.S. FDA approval be used for prostate cancer diagnosis and late period radionuclide therapy 111In-pendetide, to be connected with radionuclide based on the monoclonal antibody for PSMA exactly and to form.But compared with single domain heavy chain antibody, it is relatively high to there is production cost in these parts, the shortcomings such as preparation process is complicated.
Summary of the invention
The object of this invention is to provide the single domain heavy chain antibody for prostate specific membrane antigen, reagent and the instrument of preparing detection prostate specific membrane antigen can be used to.
The invention provides a single domain heavy chain antibody for prostate specific membrane antigen (i.e. the single domain heavy chain antibody of energy specific combination prostate specific membrane antigen of the present invention), there is the aminoacid sequence shown in SEQIDNO.:1, its aminoacid sequence is numbered the division with structural domain by standardized antibody amino acids sequence method for numbering serial (ImMunoGeneTics, IMGT).
The invention provides a kind of protein or polypeptide, it is characterized in that one or two the above aminoacid sequences comprised in framework region, and at least with an aminoacid sequence, there is 90% homology.
The invention provides a kind of protein or polypeptide, it is characterized in that one or two the above aminoacid sequences comprised in complementary determining region, and at least with an aminoacid sequence, there is 80% homology.
The invention provides a nucleic acid molecule, it is characterized in that the SEQIDNO.:1 that encodes, the concrete sequence of this nucleic acid molecule can be obtained by genetic codon at any time.The sequence of this nucleic acid molecule is as SEQIDNO.:2.
The present invention also provides a nucleic acid molecule, it is characterized in that coding SEQIDNO.:1 partial domain, can be obtained the concrete sequence of this nucleic acid molecule by genetic codon at any time.
Nucleotide sequence provided by the present invention or at least partly sequence can be undertaken expressing to obtain corresponding protein or polypeptide by suitable expression system.These expression systems comprise bacterium, yeast, filamentous fungus, zooblast, insect cell, vegetable cell, or Cell free expression system.
The present invention also provides a kind of carrier, comprises described nucleotide sequence.Because genetic codon has degeneracy, this nucleotide sequence can be different according to different application purposes.
The present invention also provides a kind of host cell, comprises described protein or expression vector.
The present invention also provides a kind of method detecting prostate specific membrane antigen on cell, it is characterized in that conjunction has above-mentioned protein or polypeptide.Based on the ability of protein provided by the invention or polypeptide and prostate specific membrane antigen specific binding, set up the detection method of prostate specific membrane antigen.Wherein, preferred method comprises enzyme-linked immunosorbent assay (Enzyme-linkedimmunosorbentassay, ELISA), fluorescent immune method (Fluoroimmunoassay, FIA), immuno-chip method, affinity chromatography and immunochromatographic method.
The present invention is directed to the application of prostate specific membrane antigen single domain heavy chain antibody in non-diseases diagnoses and treatment object immunodetection and thalline or antigen enrichment purifying.
Aminoacid sequence provided by the present invention can as precursor, transformed by random or site-directed mutagenesis technique, character (water-soluble, stability, avidity and specificity etc.) better mutant can be obtained, be used for developing being further used for medicine, industry, agriculture protein or polypeptide.
The invention still further relates to the affine sorbing material of a kind of immunity for prostate specific membrane antigen, comprise carrier, be mounted in the aglucon on carrier, this material is using the single domain heavy chain antibody for prostate specific membrane antigen as aglucon, and the described single domain heavy chain antibody for prostate specific membrane antigen has the aminoacid sequence shown in SEQIDNO.:1.Described carrier is magnetic bead, agarose gel microsphere, silica gel microball or porous material.
The present invention some terms of describing there is following implication:
Homology: the similarity degree describing two or more aminoacid sequences, between first aminoacid sequence and second aminoacid sequence, the per-cent of homology can be multiplied by [100%] to calculate divided by [in first aminoacid sequence amino acid sum] by [quantity of amino-acid residue identical with the aminoacid sequence of corresponding position in second aminoacid sequence in first aminoacid sequence], and certain the amino acid whose disappearance wherein in second aminoacid sequence, insertion, replacement or interpolation (compared with first amino acid) are considered to there is difference.Alternatively, percent homology also can utilize known Computing program such as the NCBIBlast for sequences match to obtain.
Structural domain: the fundamental structural unit of tertiary protein structure, has certain function usually.
IMGT numbers: the one in IMGT database (TheInternationalImMunoGeneTicsDatbase) is standardized antibody amino acids sequence method for numbering serial.Concrete method for numbering serial can reference (Ehrenman, F., Q.Kaas, etal. (2010). " IMGT/3Dstructure-DBandIMGT/DomainGapAlign:adatabaseandat oolforimmunoglobulinsorantibodies, Tcellreceptors, MHC, IgSFandMhcSF. " NucleicAcidsRes38 (Databaseissue): D301-307.Lefranc, M.P., C.Pommie, etal. (2003). " IMGTuniquenumberingforimmunoglobulinandTcellreceptorvari abledomainsandIgsuperfamilyV-likedomains " DevcompImmunol27 (1): 55-77.http: //www.imgt.org/) in description.
Codon (codon): be also called three disjunctor codons (tripletcode), refers to correspond to certain amino acid whose nucleotide triplet.In the process of translating, determine that this seed amino acid inserts the position of polypeptide chain in growth.
Beneficial effect of the present invention: the present invention is directed to the single domain heavy chain antibody of prostate specific membrane antigen or polypeptide have with prostate specific membrane antigen specific binding, can by biological method scale operation, the character such as cost is low, efficient, molecular weight is little, good penetrability, demonstrate good application prospect.
Accompanying drawing explanation
Fig. 1 bacterium colony PCR primer electrophoresis, recombinant protein electrophoresis and WesternBlot identify figure.Left side Marker swimming lane is DNA molecular amount standard, and the bacterium colony PCR fragment in swimming lane 1 appears at desired location.Centre is that the albumen after purifying carries out SDS-PAGE detection, occurs bright band in desired location.The right is the WesternBlot qualification figure with anti-PSMA monoclonal antibody and anti-His tag antibody.
Embodiment
Below by preparation, the Analyzes and nurses of single domain heavy chain antibody (polypeptide), the present invention will be further described, and these specific embodiments should not be interpreted as limiting range of application of the present invention by any way.
Embodiment 1
The eukaryotic expression of prostate specific membrane antigen film outskirt
RNAisoPlus reagent is adopted to extract total serum IgE in the LNCaP cell of high expression level PSMA, RT-PCR method is utilized to obtain the DNA fragmentation of coding PSMA extracellular domain fragment, use NotI and BamHI two kinds of restriction enzymes to be inserted into carrier for expression of eukaryon pRAG2a, connected for recombinant plasmid by T4DNA ligase enzyme.Recombinant plasmid thermal shock is transformed into TOP10 competent cell overnight incubation, send sequence verification by the correct clone of qualification.Extract the plasmid of positive colony, use liposome Lipofectamine2000 to be cultivated to HEK-293 cell by DNA plasmid transfection, collect supernatant in different time phases, carry out SDS-PAGE electrophoresis detection.After cultivating certain hour, according to this albumen of HisTrapFFCrude test kit operation purifying.After albumen after purifying is carried out SDS-PAGE, electricity goes on pvdf membrane, and after 5% skim-milk is closed, add PSMA antibody and His antibody respectively, 4 DEG C are spent the night; After rinsing, add two anti-incubated at room temperature 1h, rinsing again, add nitrite ion and carry out develop (Fig. 1).
Embodiment 2
The elutriation of anti-prostate specific membrane antigen single domain heavy chain antibody (namely for anti-prostate specific membrane antigen single domain heavy chain antibody) and qualification
The method of solid phase elutriation is adopted (to be that reference: ＂ painting chases after from hunchbacked source natural antibody phage display library, Xu Yang, Liu Xia, Deng. the Construction and identification [J] in natural single domain heavy chain antibody storehouse, hunchbacked source. Chinese biological engineering magazine, the display libraries built in 2011,31 (4): 31-36. ＂) in elutriation for the single domain heavy chain antibody of prostate specific membrane antigen.The expression of prostate specific membrane antigen film outskirt is carried out according to the above embodiments 1, during first round elutriation, adopt phosphate buffer soln (PBS, pH7.4) PSMA extracellular region protein to the 150 μ g/mL (2-4 wheel bag is respectively 100,50,50 μ g/mL by concentration) of above-mentioned synthesis is diluted, on enzyme plate, every hole adds 100 μ L, and 4 DEG C of bags are spent the night.PBST (closing 0.5%Tween20) washes plate 5 times, and 3%BSA-PBS, at 37 DEG C of closed 2h, washes plate 3 times, adds the phage antibody library 100 μ L of hatching with 0.5%BSA-PBS and (is roughly equal to 2 × 10 11cFU), hatch 1h for 37 DEG C, wash plate 5 times (by wheel increase by 3 times) with PBST, then wash plate 10 times (by wheel increase by 5 times) with PBS.Again with 100 μ L elutriant (glycine-HCI, pH2.2) (37 DEG C, the phage of wash-out absorption, 5min), with 50 μ LTris-HCl (1mol/L, pH9.0) in and eluate, get 10 μ L for titer determination, for next round elutriation after the amplification of all the other eluates.
After four-wheel elutriation, adopt concentration to be the PSMA extracellular region protein coated elisa plate of 5 μ g/mL, wash plate, close the same.The phage clone 37 DEG C adding amplification purification hatches 15min, and 100 μ L/ holes, hatch 1h for 37 DEG C.Add the HRP-anti-M13 antibody that extent of dilution is 1:5000 after washing plate, 100 μ L/ holes, hatch 1h for 37 DEG C.PBST washes plate 5 times, adds TMB working fluid 100 μ L/ hole, room temperature 20min, and every hole adds 50 μ L sulfuric acid (concentration is 2mol/L) termination reaction, measures 450nm light absorption value.Using the phage antibody library direct coated enzyme plate of previous round amplification as positive control, substitute phage clone for blank with PBS, measure the binding activities of phage particle by indirect phage-ELISA method.
Table 1 is phage-ELISA application of sample table indirectly
Send order-checking company to carry out sequencing ELISA positive colony, obtain the Insert Fragment DNA sequence dna of anti-prostate specific membrane antigen single domain heavy chain antibody phage positive colony, be specially (SEQIDNO.:2):
The aminoacid sequence of its coding of ATGGCCCAGTTGCAGCTCGTGGAGTCAGGGGGAGGCTTGGTGCAGCCTGGGGGGTC CCTAAGACTCACCTGTACAACCTCTGGATTCAGTTTGAGATATCATGACATCGGTT GGTTCCGCCAGGCCCCAGGGAAGGAGCGCGAGGGGGTCTCATGTATTACTGGGAGC CTCGGTGACCCCTACGTTGACGAGTCCCTACAGGGCCGATTCACCATCTCCAGAGA CGACCACAAGAATATGGTGTATCTGCGAATGAACAGCCTGAAATCTGAGGACACGG CCCTTTATTATTGCGCGATAGTGCGCGGGTGCAAGGACCATGACGCGTACGCAGAC GTGCGAGCCCGGGGCCAGGGGACGCAGGTCACCGTCTCCTCGGAACCCAAGACACC AAAACCACAAGCGGCCGCA is as shown in SEQIDNO.:1:
QLQLVESGGGLVQPGGSLRLTCTTSGFSLRYHDIGWFRQAPGKEREGVSCITGSLGDPYVDESLQGRFTISRDDHKNMVYLRMNSLKSEDTALYYCAIVRGCKDHDAYADVRARGQGTQVTVSS。The i.e. single domain heavy chain antibody of the anti-prostate specific membrane antigen of the present invention, its can with the film outskirt generation specific binding of psma protein.
Embodiment 3
The ELISA of PSMA express cell and fluorescence immunoassay detect
Gastric cancer cell MKN45 does not express PSMA, and prostate cancer cell LNCaP expresses PSMA, for these two kinds of cells, the anti-prostate specific membrane antigen single domain heavy chain antibody phage positive colony of elutriation acquisition in embodiment 2 is adopted to carry out ELISA and the fluorescence immunoassay detection of cell levels.Plant respectively in 96 orifice plates into LNCaP cell (expressing PSMA) and MKN45 cell (not expressing PSMA) each 1 × 10 4individual, overnight incubation.4% paraformaldehyde is fixed, and every hole drips the 3% hydrogen peroxide liquid of 100 μ L, in order to block endogenous peroxidase activity, hatches 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 fluid and OD 450mensuration with embodiment 2.Positive control uses phage to substitute cell, and blank uses PBS to substitute the phage clone of interpolation, repeats 3 times.
Plant into LNCaP cell and MKN45 cell each 4 × 10 add cell climbing sheet in the every hole in 24 orifice plates after 5individual, overnight incubation.Take out creep plate, 4% paraformaldehyde is fixed, 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 the phage clone of nano antibody of the present invention hatches 1h, not add the cell climbing sheet of phage clone for blank.Thereafter add extent of dilution be 1: 2000 anti-M13 monoclonal antibody hatch 30min.Drip after developing a film 30 μ l extent of dilution be 1: 200 FITC-goat anti-mouse two resist, lucifuge hatches 30min, and redyes with DAPI staining fluid, is placed in fluorescence microscopy Microscopic observation.
Result shows: because the MKN45 cell measured value of not expressing PSMA there are differences with blank statistically, show this phage can with its generation non-specific binding; But LNCaP cell measured value is significantly higher than MKN45 cell, show that this part variation comes from the specific binding of PSMA and anti-prostate specific membrane antigen single domain heavy chain antibody phage positive colony.Simultaneously, cellular immunofluorescence shows that LNCaP cell can be combined with the single domain heavy chain antibody positive colony for prostate specific membrane antigen, and MKN45 cell and do not add anti-prostate specific membrane antigen single domain heavy chain antibody phage positive colony cell climbing sheet for negative, show that anti-prostate specific membrane antigen single domain heavy chain antibody phage positive colony that elutriation goes out can express the cell generation specific binding of the positive effectively with PSMA.
Embodiment 4
The expression of single domain heavy chain antibody in intestinal bacteria of anti-prostate specific membrane antigen
The phagemid extracted for the single domain heavy chain antibody positive colony of prostate specific membrane antigen in embodiment 2 is template, design primer amplified goal gene, amplification condition: 94 DEG C of 5min denaturations; 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 40s totally 30 circulations; Last 72 DEG C extend 5min again.Detect with 1% agarose gel electrophoresis after terminating, and cut glue recovery object fragment.
The double digestion gene fragment clone of the single domain heavy chain antibody of the anti-prostate specific membrane antigen provided by the present invention that obtains encoding is connected to expression vector pET-28a, after sequence verification, obtains recombinant plasmid.
Recombinant plasmid transformed is in e. coli bl21 (Rosseta), and picking list bacterium colony carries out abduction delivering.By single colony inoculation in the test tube of LB substratum, 37 DEG C of shaking culture, activated overnight; Next day, the ratio in 1% is transferred in fresh LB liquid nutrient medium, and 37 DEG C, 250rpm shaking culture, to OD 600after being about 0.6, add the IPTG that final concentration is 0.1mM, 37 DEG C, 250rpm induces 4h.
The 2mL bacterium liquid of above-mentioned induction obtains thalline by 8000rpm is centrifugal, thalline uses aseptic PBS to wash 3 times, and carry out resuspended thalline with the aseptic PBS of 1mL, ultrasonication thalline is until bacterium liquid is limpid on ice, carries out centrifugal at 4 DEG C to cell pyrolysis liquid, and centrifugal condition is 12000rmp/min, time is 10min, get supernatant and add 5 μ l5 × SDS sample-loading buffers, boiling water boils 5min, gets supernatant liquor and carry out SDS-PAGE electrophoretic analysis and adopt nickel post to carry out purifying to it after centrifugal.
By optimizing abduction delivering condition (as Host Strains, expression vector, induction time, inducing temperature and IPTG concentration etc.), the expression amount of target protein (single domain heavy chain antibody) can be improved further, for the single domain heavy chain antibody preparing anti-prostate specific membrane antigen in a large number provides approach.
Embodiment 5
For the mensuration of the single domain heavy chain antibody affinity costant of prostate specific membrane antigen
Adopt biotinylation kit to carry out biotinylation mark the single domain heavy chain antibody of expressing in embodiment 4, use thereafter the competitive ELISA technical measurement biotinylation of standard for psma protein affinity recombinant expressed in the single domain heavy chain antibody of prostate specific membrane antigen and embodiment 1.Concrete steps are: first adopt the biotinylated single domain heavy chain antibody for prostate specific membrane antigen of 1nM in EP pipe, to carry out 30min with the PSMA antigen of 13 kinds of different concns (0.1nM ~ 100 μM) respectively and hatch; Thereafter, 90 μ L mixed solutions are added use that 3%BSA-PBST closes, be coated with in the enzyme plate of psma protein, after hatching 10min, inhale and abandon reaction solution, and to clean with PBST; Then, add the Streptavidin that 100 μ L extent of dilution are 1:2000HRP mark, after hatching 1h, adopt PBST to clean 5 times; The use of TMB working fluid and OD 450mensuration with embodiment 2.Nonlinear regression analysis is adopted to obtain OD 450maximum value one half, according to the principle of Ag-Ab competitive binding experiment, corresponding PSMA concentration, show that the biotinylated single domain heavy chain antibody affinity costant for prostate specific membrane antigen is 5 × 10 -7about/M.
The preparation of the immune affine sorbing material of embodiment 6
1, the preparation of immune affine magnetic bead
Adopt nanometer magnetic bead as carrier, coupling is for after the single domain heavy chain antibody of prostate specific membrane antigen, and obtain the single domain heavy chain antibody immunomagnetic beads for prostate specific membrane antigen, concrete preparation method is as follows:
The magnetic bead (transporting nanosecond science and technology company limited purchased from Wuxi hundred, carboxyl magnetic bead 300nm) getting 1mg carboxyl modified, in centrifuge tube, adds 500 μ l activation buffer (10mM, NaH 2pO 4, pH6.0), vortex mixed is even, and magnetic frame reclaims magnetic bead, then washs 2 times with activation buffer.Add 2mg carbodiimide (EDC) and N-hydroxy-succinamide (NHS) respectively, after vortex mixed, leave standstill 30min.With coupling buffer (10mM, Na 2hPO 4pH7.4) magnetic bead is washed 3 times, add the single domain heavy chain antibody 1mg for prostate specific membrane antigen being dissolved in coupling buffer, room temperature reaction 3h, magnetic bead is washed 3 times with coupling buffer, the coupling buffer adding 500 μ l conjunction 1% (w/v) bovine serum albumins (BSA) or 1% (w/v) fourth of the twelve Earthly Branches white protein (OVA) closes unreacted active group, room temperature reaction 30min.Magnetic bead is washed 3 times, PBS solution (10mM, pH7.4,0.02%w/v, Na with coupling buffer 3n) 4 DEG C are stored in after resuspended.
2, for the single domain heavy chain antibody affine sorbing material of immunity of prostate specific membrane antigen and the preparation of affinity column.Adopt 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 0.1MHC1 that CNBr activates is washed 10 times, balances 5min at every turn.With coupling buffer (10mM, Na 2hPO 4pH7.4) wash 10 times, add the single domain heavy chain antibody (2mg/ every gram agarose microbeads) for prostate specific membrane antigen, room temperature reaction 4h, make the agarose gel microsphere covalent coupling that single domain heavy chain antibody and CNBr for prostate specific membrane antigen activate.With coupling buffer (10mM, Na 2hPO 4, pH7.4) wash 2 times after, add confining liquid room temperature reaction 2h with close unreacted active group.The phosphoric acid buffer (10mM, pH7.4) long-pending with 5 times of colloids and acetate buffer solution (0.1M, pH4.0) replace washing 3 times, obtain the immunity affine sorbing material of covalent coupling for the single domain heavy chain antibody of prostate specific membrane antigen.Getting the affine sorbing material of the above-mentioned immunity of 0.2ml is the chromatography column of 1ml in capacity, after PBS (10mM, the pH7.4) washing of 5 ~ 10 times of column volumes, adds 20% ethanolic soln, 4 DEG C of preservations.
3, for the single domain heavy chain antibody affine sorbing material of immunity of prostate specific membrane antigen and the preparation of affinity column.Adopt 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:
Get 2g silica gel microball pure water and phosphoric acid buffer (PBS, 10mM, pH6.0) washing 5 ~ 10 times is replaced, with 10mlPBS damping fluid suspension silica gel microball, add the single domain heavy chain antibody of 5mg for prostate specific membrane antigen, mixing, add the carbodiimide (EDC) of final concentration 5mg/ml, rapid mixing, 4 DEG C of stirring reaction 12 ~ 24h, obtain the immunity affine sorbing material of covalent coupling for the single domain heavy chain antibody of prostate specific membrane antigen.Getting the affine sorbing material of the above-mentioned immunity of 0.2ml is the chromatography column of 1ml in capacity, after PBS (10mM, the pH6) washing of 5 ~ 10 times of column volumes, adds conjunction 0.02% (w/v) Na 3the PBS (10mM, pH6) of N, 4 DEG C of preservations.
The affine sorbing material aglucon of immunity that the present invention is directed to prostate specific membrane antigen is single domain heavy chain antibody, and have the aminoacid sequence shown in SEQIDNO.:1, this aglucon can specific recognition prostate specific membrane antigen.This single domain heavy chain antibody easily obtains, adsorption efficiency is high, and can produce aglucon by biological method mass propgation is single domain heavy chain antibody, avoids the loaded down with trivial details production methods such as artificial antibody, greatly reduces production cost.Have acid and alkali-resistance, high temperature resistant and be easy to the characteristics such as production, these characteristics have important practical value for the low cost of prostate specific membrane antigen, reusable purifying and immunological detection method.

Claims (9)

1., for the single domain heavy chain antibody of prostate specific membrane antigen, there is the aminoacid sequence shown in SEQIDNO.:1.
2. a nucleic acid molecule, is characterized in that aminoacid sequence described in coding claim 1.
3. nucleic acid molecule according to claim 2, is characterized in that sequence is as SEQIDNO.:2.
4. one kind comprises the carrier of nucleotide sequence according to claim 2.
5. one kind comprises the host cell of carrier according to claim 4.
6. according to claim 1 for the application of prostate specific membrane antigen single domain heavy chain antibody in immunodetection, thalline or antigen enrichment purifying.
7. the application of single domain heavy chain antibody in preparation prostate specific membrane antigen immunodetection, enrichment and purified reagent or material for prostate specific membrane antigen according to claim 1.
8. the single domain heavy chain antibody for prostate specific membrane antigen according to claim 1 by random or site-directed mutagenesis technique carry out house of correction acquisition can with the antibody of prostate specific membrane antigen specific binding.
9. the affine sorbing material of the immunity for prostate specific membrane antigen, comprise carrier, be mounted in the aglucon on carrier, it is characterized in that this material is using the single domain heavy chain antibody for prostate specific membrane antigen as aglucon, the described single domain heavy chain antibody for prostate specific membrane antigen has the aminoacid sequence shown in SEQIDNO.:1.
CN201610073980.7A 2016-02-03 2016-02-03 Single domain heavy-chain antibody capable of being specifically bound with prostate specific membrane antigen Pending CN105542008A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101124249A (en) * 2005-02-18 2008-02-13 米德列斯公司 Human monoclonal antibodies to prostate specific membrane antigen(PSMA)
CN103497252A (en) * 2013-09-22 2014-01-08 南昌大学 Single-domain heavy chain antibody L5-78 for Listeria monocytogenes

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101124249A (en) * 2005-02-18 2008-02-13 米德列斯公司 Human monoclonal antibodies to prostate specific membrane antigen(PSMA)
CN103497252A (en) * 2013-09-22 2014-01-08 南昌大学 Single-domain heavy chain antibody L5-78 for Listeria monocytogenes

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范校周等: "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", 《第三军医大学学报》 *

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