CN105622755A - Nano antibody of anti-prostate specific membrane antigen extracellular region - Google Patents

Nano antibody of anti-prostate specific membrane antigen extracellular region Download PDF

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CN105622755A
CN105622755A CN201610074011.3A CN201610074011A CN105622755A CN 105622755 A CN105622755 A CN 105622755A CN 201610074011 A CN201610074011 A CN 201610074011A CN 105622755 A CN105622755 A CN 105622755A
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membrane antigen
specific membrane
prostate specific
heavy chain
single domain
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CN105622755B (en
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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 the field of genetic engineering, and a single-domain heavy-chain antibody aiming at prostate specific membrane antigen. The antibody has an amino acid sequence shown as SEQ ID NO.:1 and can be used in the field of immunodetection and antigen enrichment and purification. The amino acid sequence can serve as a precursor, mutants with higher properties (like compatibility, specificity and stability) can be obtained by modifying through random or fixed-point mutation technology, and the antibody is used for developing protein or polypeptide further being used for medicine, industry and agriculture.

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 technology, especially 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 being 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 being only made up of is referred to heavy chain antibody (heavy-chainantibody) variable region (Variableregion), wherein, heavy chain antibody (heavy-chainantibody) is a kind of to be present in the animal body such as camel, shark the antibody of natural deletions light chain. Single domain heavy chain antibody is the minimum intact antigen binding fragment being currently known, and has the features such as little, the good penetrability of molecular weight, is widely used in basic research, medical diagnosis and the field such as detection, antibody drug exploitation at present.
Carcinoma of prostate is the malignant tumor that a kind of serious threat elderly men is healthy, and its early diagnosis and therapy has great importance for its prognosis. prostate specific membrane antigen (prostatespecificmembraneantigen, PSMA) it is a kind of II type transmembrane glycoprotein being positioned on prostatic cell film, it is made up of 750 aminoacid, it is divided into intracellular region (aminoacid sequence is 1-18), cross-film district (19-43) and extracellular region (44-750), relative to the prostate specific antigen (prostatespecificantigen being conventionally used to Clinical detection, PSA), it it is a kind of more sensitive and special prostate cancer label, it is high expressed particularly in hormone-refractory prostate cancer and carcinoma of prostate metastasis, distinguishing the sensitivity of carcinoma of prostate and other types malignant tumor and specificity respectively 65.9% and 94.5%. it addition, at the solid tumor in multiple non-prostate source, such as pulmonary carcinoma, bladder cancer, gastric cancer, cancer of pancreas, renal carcinoma and colorectal cancer etc., be expressed on tumor vascular endothelial cell PSMA also high special. and itself there is neural carboxypeptidase and the activity of folic acid hydrolytic enzyme, the extracellular region of 707 aminoacid compositions can provide the features such as multiple epitopes in addition so that it is becomes research target spot important in tumour immunity targeted therapy and molecular imaging.
At present, it is found that multiple being also prepared for for PSMA multiple specific binding material can occur with it, including monoclonal antibody 7E11, J591, fit A9 and A10, the report of ScFv antibody D7, Fab antibody and humanized antibody that recombinant technique obtains, particularly already by the U.S. FDA approval diagnosis for carcinoma of prostate and late period radionuclide therapy 111In-pendetide, it is simply that be connected with radionuclide based on the monoclonal antibody for PSMA and form. But compared with single domain heavy chain antibody, it is of a relatively high to there is production cost in these parts, the shortcomings such as preparation process is complicated.
Summary of the invention
It is an object of the invention to provide the single domain heavy chain antibody for prostate specific membrane antigen, it is possible to be used to reagent and the instrument of preparation detection prostate specific membrane antigen.
The present invention provides a single domain heavy chain antibody for prostate specific membrane antigen (i.e. the nano antibody of a kind of anti-prostate-specific membrane antigen extracellular region of the present invention), there is the aminoacid sequence shown in SEQIDNO.:1, its aminoacid sequence can pass through standardized antibody amino acids sequence method for numbering serial (ImMunoGeneTics, IMGT) and be numbered the division with domain.
The present invention provides a kind of protein or polypeptide, it is characterized in that one or two the above aminoacid sequences comprising in framework region, and at least has 90% homology with an aminoacid sequence.
The present invention provides a kind of protein or polypeptide, it is characterized in that one or two the above aminoacid sequences comprising in complementary determining region, and at least has 80% homology with an aminoacid sequence.
The present invention provides a nucleic acid molecules, it is characterized in that coding SEQIDNO.:1, can be obtained the particular sequence of this nucleic acid molecules by genetic codon at any time. The sequence of this nucleic acid molecules such as SEQIDNO.:2.
The present invention also provides for a nucleic acid molecules, it is characterized in that coding SEQIDNO.:1 partial domain, can be obtained the particular sequence of this nucleic acid molecules 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 include antibacterial, yeast, filamentous fungi, zooblast, insect cell, plant cell, or Cell free expression system.
The present invention also provides for a kind of carrier, comprises described nucleotide sequence. Owing to genetic codon has degeneracy, this nucleotide sequence can be different according to different application purposes.
The present invention also provides for a kind of host cell, including described protein or expression vector.
The present invention also provides for a kind of detecting the method for prostate specific membrane antigen on cell, it is characterized in that containing above-mentioned protein or polypeptide. Based on the ability that protein provided by the invention or polypeptide and prostate specific membrane antigen are specific binding, set up the detection method of prostate specific membrane antigen. Wherein, it is preferred that method includes 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 in non-diseases diagnoses and treatment purpose immune detection and thalline or antigen enrichment purification of the prostate specific membrane antigen single domain heavy chain antibody.
Aminoacid sequence provided by the present invention can as precursor, transformed by random or site-directed mutagenesis technique, it is obtained in that character (water solublity, stability, affinity and specificity etc.) better mutant, is further used for medicine, industry, agriculture protein or polypeptide for developing.
The invention still further relates to a kind of affine adsorbing material of the immunity for prostate specific membrane antigen, including carrier, it is 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.
Some terms that the present invention describes have following implication:
Homology: describe the similarity degree of two or more aminoacid sequences, between first aminoacid sequence and second aminoacid sequence, the percentage ratio of homology can pass through [quantity of amino acid residue identical with the aminoacid sequence of corresponding position in second aminoacid sequence in first aminoacid sequence] and calculates being multiplied by [100%] divided by [in first aminoacid sequence aminoacid sum], wherein certain the amino acid whose disappearance in second aminoacid sequence, insertion, replacement or to add (compared with first aminoacid) be considered as have difference. Alternatively, percent homology can also utilize the known Computing program such as NCBIBlast for sequences match to obtain.
Domain: the fundamental structural unit of tertiary protein structure, is generally of certain function.
IMGT numbers: the one in IMGT data base (TheInternationalImMunoGeneTicsDatbase) is standardized antibody amino acids sequence method for numbering serial. concrete method for numbering serial is referred to document (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), refer to corresponding to certain amino acid whose nucleotide triplet. Translation process determining, this seed amino acid inserts the position of polypeptide chain in growth.
Beneficial effects of the present invention: the present invention is directed to the single domain heavy chain antibody of prostate specific membrane antigen or polypeptide have specific binding with prostate specific membrane antigen, the character such as biological method large-scale production, cost is low, efficient, molecular weight is little, good penetrability can be passed through, it is shown that good application prospect.
Accompanying drawing explanation
Fig. 1 bacterium colony PCR primer electrophoresis, recombiant 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 occurs in desired location. Centre carries out SDS-PAGE detection for the albumen after purification, bright band occurs in desired location. The right is identify figure with the WesternBlot of anti-PSMA monoclonal antibody and anti-His tag antibody.
Detailed description of the invention
Below by the preparation of single domain heavy chain antibody (polypeptide), analysis and application, the present invention will be further described, and these specific embodiments are not construed in any way as limiting the range of application of the present invention.
Embodiment 1
The eukaryotic expression of prostate specific membrane antigen film outskirt
RNAisoPlus reagent is adopted to extract total serum IgE in the LNCaP cell of high expressed PSMA, RT-PCR method is utilized to obtain the DNA fragmentation of coding PSMA extracellular domain fragment, use two kinds of restricted enzyme of NotI and BamHI to be inserted into carrier for expression of eukaryon pRAG2a, be connected as recombiant plasmid by T4DNA ligase. Recombiant plasmid thermal shock is transformed into TOP10 competent cell overnight incubation, will identify that correct clone send sequence verification. Extract the plasmid of positive colony, use liposome Lipofectamine2000 by DNA plasmid transfection to HEK-293 cell is cultivated, collect supernatant in different time phases, carry out SDS-PAGE electrophoresis detection. After cultivating certain time, operate this albumen of purification according to HisTrapFFCrude test kit. After albumen after purification is carried out SDS-PAGE, electricity goes on pvdf membrane, and 5% defatted milk powder is separately added into PSMA antibody and His antibody after closing, and 4 DEG C overnight; After rinsing, the anti-incubated at room temperature 1h that adds two, again rinse, add nitrite ion and carry out develop (Fig. 1).
Embodiment 2
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
Adopt the method for solid phase elutriation to be coated with and chase after from camel source natural antibody phage display library (for list of references: "; Xu Yang; Liu Xia; etc. the structure in camel natural single domain heavy chain antibody storehouse, source with identify [J]. Chinese biological engineering magazine; 2011,31 (4): 31-36. " in the display libraries that builds) in elutriation for the single domain heavy chain antibody of prostate specific membrane antigen. The expression of prostate specific membrane antigen film outskirt carries out according to the above embodiments 1, during first round elutriation, adopt phosphate buffered solution (PBS, pH7.4) the PSMA extracellular region protein of above-mentioned synthesis is diluted to 150 �� g/mL (2-4 wheel is coated concentration respectively 100,50,50 �� g/mL), in ELISA Plate, every hole adds 100 �� L, and 4 DEG C are coated overnight. PBST (containing 0.5%Tween20) washes plate 5 times, and 3%BSA-PBS closes 2h at 37 DEG C, washes plate 3 times, adds the phage antibody library 100 �� L hatched with 0.5%BSA-PBS (containing about 2 �� 1011CFU), hatch 1h for 37 DEG C, wash plate 5 times (increasing by 3 times by wheel) with PBST, then wash plate 10 times (increasing by 5 times by wheel) with PBS. Again with 100 �� L eluent (glycine-HCI, pH2.2) phage (37 DEG C of eluting absorption, 5min), with 50 �� LTris-HCl (1mol/L, pH9.0) eluate is neutralized, take 10 �� L for titer determination, for next round elutriation after all the other eluates amplification.
The PSMA extracellular region protein coated elisa plate adopting concentration to be 5 �� g/mL after four-wheel elutriation, washes plate, closes ibid. The phage clone 37 DEG C adding amplification purification hatches 15min, and 1h is hatched for 37 DEG C in 100 �� L/ holes. Add the HRP-anti-M13 antibody that dilution factor is 1: 5000,100 �� L/ holes after washing plate, hatch 1h for 37 DEG C. PBST washes plate 5 times, adds TMB working solution 100 �� L/ hole, room temperature 20min, and every hole adds 50 �� L sulphuric acid (concentration is 2mol/L) and terminates reaction, measures 450nm light absorption value. Using the phage antibody library direct coated ELISA Plate of previous round amplification as positive control, substitute phage clone for blank with PBS, measure the combination activity of phage particle by indirect phage-ELISA method.
The indirect phage-ELISA application of sample table of table 1
Send order-checking company to carry out sequencing ELISA positive colony, obtain the Insert Fragment DNA sequence of anti-prostate specific membrane antigen single domain heavy chain antibody phage positive colony, be specially (SEQIDNO.:2):
ATGGCCCAGGTGCAGCTCGTGGAGTCGGGGGGAGGCTTGGTGCAGGCTGGGGATTCTCTGAGACTCTCCTGTGCAGCCTCTAGAAGGACGTTCAGTTTCATGGCATGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGCAGCTATTACTAGTGGTGGTCGTAATAC AAACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACGATGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTACTGTAACGCGGCGGCCCGGTGGAATAACTACTGGGGCCAGGGGACCCCGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAGCGGCCGC
The aminoacid sequence of its coding is such as shown in SEQIDNO.:1:
QVQLVESGGGLVQAGDSLRLSCAASRRTFSFMAWYRQAPGKQRELVAAITSGGRNT NYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCNAAARWNNYWGQGTPVTV SS. The i.e. single domain heavy chain antibody of the anti-prostate specific membrane antigen of the present invention, it can occur specific binding with the film outskirt of psma protein.
Embodiment 3
The ELISA of PSMA express cell and fluorescence immunoassay detection
Gastric cancer cell MKN45 does not express PSMA, and prostate gland cancer cell LNCaP expresses PSMA, for both cells, the anti-prostate specific membrane antigen single domain heavy chain antibody phage positive colony that in embodiment 2, elutriation obtains is adopted to carry out ELISA and the fluorescence immunoassay detection of cellular level. Plant respectively in 96 orifice plates into LNCaP cell (expressing PSMA) and MKN45 cell (not expressing PSMA) each 1 �� 104Individual, overnight incubation. 4% paraformaldehyde is fixed, and every hole drips the 3% hydrogen peroxide liquid of 100 �� 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 �� L anti-prostate specific membrane antigen single domain heavy chain antibody phage positive colony, hatches 1h in 37 DEG C. Thereafter PBS rinsing, adds HRP-anti-M13 antibody, TMB working solution and OD450Mensuration with embodiment 2. Positive control uses phage to substitute cell, and blank uses PBS to substitute the phage clone added, and repeats 3 times.
Plant into LNCaP cell and MKN45 cell each 4 �� 10 after adding cell climbing sheet in the every hole in 24 orifice plates5Individual, overnight incubation. Taking 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 �� L show nano antibodies of the present invention phage clone hatch 1h, be not added with phage clone cell climbing sheet for blank. Thereafter add the anti-M13 monoclonal antibody that dilution factor is 1: 2000 and hatch 30min. Dripping the FITC-goat anti-mouse two that 30 �� l dilution factors are 1: 200 after developing a film to resist, lucifuge hatches 30min, and redyes with DAPI dyeing liquor, is placed in fluorescence microscopy Microscopic observation.
Result shows: the MKN45 cell measured value owing to not expressing PSMA there are differences with blank statistically, it was shown that non-specific binding can occur this phage with it; But LNCaP cell measured value is significantly higher than MKN45 cell, it was shown 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 the cell climbing sheet of anti-prostate specific membrane antigen single domain heavy chain antibody phage positive colony for feminine gender, it was shown that the anti-prostate specific membrane antigen single domain heavy chain antibody phage positive colony that elutriation goes out effectively can be expressed positive cell with PSMA and be occurred specific binding.
Embodiment 4
The expression in escherichia coli of the single domain heavy chain antibody of anti-prostate specific membrane antigen
In extraction embodiment 2, the phasmid for the single domain heavy chain antibody positive colony of prostate specific membrane antigen is template, designs primer amplified genes of interest, 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 re-extend 5min. Detect with 1% agarose gel electrophoresis after end, and cut glue recovery purpose fragment.
The double digestion gene fragment clone obtaining encoding the single domain heavy chain antibody of anti-prostate specific membrane antigen provided by the present invention is connected to expression vector pET-28a, after sequence verification, obtains recombiant plasmid.
Recombinant plasmid transformed is in e. coli bl21 (Rosseta), and picking list bacterium colony carries out abduction delivering. By single colony inoculation in the test tube of LB culture medium, 37 DEG C of shaken cultivation, activated overnight; Next day, transferring in fresh LB fluid medium in the ratio of 1%, 37 DEG C, 250rpm shaken cultivation, to OD600After being about 0.6, add the IPTG of final concentration of 0.1mM, 37 DEG C, 250rpm induce 4h.
The 2mL bacterium solution of above-mentioned induction obtains thalline by 8000rpm is centrifugal, thalline uses aseptic PBS to wash 3 times, and carry out resuspended thalline with the aseptic PBS of 1mL, ultrasonication thalline is until bacterium solution is limpid on ice, at 4 DEG C, cell pyrolysis liquid is centrifuged, and centrifugal condition is 12000rmp/min, time is 10min, taking supernatant and add 5 �� l5 �� SDS sample-loading buffer, boiling water boils 5min, takes supernatant and carry out SDS-PAGE electrophoretic analysis and adopt nickel post that it is purified after centrifugal.
By optimizing abduction delivering condition (such as Host Strains, expression vector, induction time, inducing temperature and IPTG concentration etc.), can improving the expression of destination protein (single domain heavy chain antibody) further, the single domain heavy chain antibody for preparing anti-prostate specific membrane antigen in a large number provides approach.
Embodiment 5
Mensuration for the single domain heavy chain antibody affinity costant of prostate specific membrane antigen
Adopt biotinylation kit to carry out biotinylation labelling the single domain heavy chain antibody expressed in embodiment 4, use thereafter the competitive ELISA technical measurement biotinylation of standard for the single domain heavy chain antibody of the prostate specific membrane antigen psma protein affinity recombinant expressed with embodiment 1. Concretely comprise the following steps: in EP pipe, carry out 30min with the PSMA antigen of 13 kinds of variable concentrations (0.1nM��100 ��M) respectively initially with the biotinylated single domain heavy chain antibody for prostate specific membrane antigen of 1nM and hatch; Thereafter, 90 �� L mixed liquors are added and has used in ELISA Plate that 3%BSA-PBST closes, that be coated with psma protein, after hatching 10min, inhale and abandon reactant liquor, and clean with PBST; Then, add the Streptavidin that 100 �� L dilution factors are 1: 2000HRP labelling, after hatching 1h, adopt PBST to clean 5 times; The use of TMB working solution and OD450Mensuration with embodiment 2. Nonlinear regression analysis is adopted to obtain OD450During maximum half, corresponding PSMA concentration, show that the biotinylated single domain heavy chain antibody affinity costant for prostate specific membrane antigen is 5 �� 10 according to the principle of Ag-Ab competitive binding experiment-7About/M.
The preparation of the immune affine adsorbing material of embodiment 6
1, the preparation of immune affine magnetic bead
Adopting nanometer magnetic bead as carrier, coupling is for, after the single domain heavy chain antibody of prostate specific membrane antigen, obtaining the single domain heavy chain antibody immunomagnetic beads for prostate specific membrane antigen, and concrete preparation method is as follows:
Take the magnetic bead (transporting nanosecond science and technology company limited, carboxyl magnetic bead 300nm purchased from Wuxi hundred) of 1mg carboxyl modified in centrifuge tube, add 500 �� l activation buffer (10mM, NaH2PO4, pH6.0), vortex mixed is uniform, and magnetic frame reclaims magnetic bead, then washs 2 times with activation buffer. It is separately added into 2mg carbodiimide (EDC) and N-hydroxy-succinamide (NHS), after vortex mixed, stands 30min. With coupling buffer (10mM, Na2HPO4PH7.4) washing magnetic bead 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, add the 500 �� l coupling buffer containing 1% (w/v) bovine serum albumin (BSA) or 1% (w/v) ovalbumin (OVA) and close 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 buffer3N) it is stored in 4 DEG C after resuspended.
2, for the preparation of the single domain heavy chain antibody affine adsorbing material of immunity of prostate specific membrane antigen and affinity column. Adopting agarose microbeads as carrier, the single domain heavy chain antibody of coupling prostate specific membrane antigen, concrete preparation method is as follows:
The CNBr dry glue activated 0.1MHCl is washed 10 times, balances 5min every time. With coupling buffer (10mM, Na2HPO4PH7.4) washing 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 of the single domain heavy chain antibody for prostate specific membrane antigen and CNBr activation. With coupling buffer (10mM, Na2HPO4, pH7.4) wash 2 times after, add confining liquid room temperature reaction 2h to close unreacted active group. Alternately washing 3 times of the phosphate buffer (10mM, pH7.4) long-pending with 5 times of colloids and acetate buffer solution (0.1M, pH4.0), obtain the covalent coupling affine adsorbing material of immunity for the single domain heavy chain antibody of prostate specific membrane antigen. Taking the 0.2ml affine adsorbing material of above-mentioned immunity is the chromatographic column of 1ml in capacity, after PBS (10mM, the pH7.4) washing of 5��10 times of bed volumes, adds 20% alcoholic solution, 4 DEG C of preservations.
3, for the preparation of the single domain heavy chain antibody affine adsorbing material of immunity of prostate specific membrane antigen and 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:
Take 2g silica gel microball pure water and phosphate buffer (PBS, 10mM, pH6.0) washing 5��10 times is replaced, with 10mlPBS buffer suspension silica gel microball, add the 5mg single domain heavy chain antibody for prostate specific membrane antigen, mixing, add the carbodiimide (EDC) of final concentration 5mg/ml, mixing, 4 DEG C of stirring reaction 12��24h, obtain the covalent coupling affine adsorbing material of immunity for the single domain heavy chain antibody of prostate specific membrane antigen rapidly. Taking the 0.2ml affine adsorbing material of above-mentioned immunity is the chromatographic column of 1ml in capacity, after PBS (10mM, the pH6) washing of 5��10 times of bed volumes, adds containing 0.02% (w/v) Na3The PBS (10mM, pH6) of N, 4 DEG C of preservations.
The present invention is directed to the affine adsorbing material aglucon of the immunity of prostate specific membrane antigen is single domain heavy chain antibody, has the aminoacid sequence shown in SEQIDNO.:1, and this aglucon can specific recognition prostate specific membrane antigen. This single domain heavy chain antibody is readily available, adsorption efficiency is high, it is possible to producing aglucon by biological method mass propgation is single domain heavy chain antibody, it is to avoid the loaded down with trivial details production methods such as artificial antibody, greatly reduces production cost. There is acid and alkali-resistance, high temperature resistant and the characteristic such as be readily produced, these characteristics have important practical value for the low cost of prostate specific membrane antigen, reusable purification 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 molecules, is characterized in that aminoacid sequence described in coding claim 1.
3. nucleic acid molecules according to claim 2, it is characterised in that sequence is SEQIDNO.:2 such as.
4. the carrier of the nucleotide sequence comprised described in claim 2.
5. the host cell of the carrier comprised described in claim 4.
6. described in claim 1 for the application in immune detection, thalline or antigen enrichment purification of the prostate specific membrane antigen single domain heavy chain antibody.
7. the application in preparation prostate specific membrane antigen immune detection, enrichment and purified reagent or material of the single domain heavy chain antibody for prostate specific membrane antigen described in claim 1.
8. described in claim 1 for prostate specific membrane antigen single domain heavy chain antibody by random or site-directed mutagenesis technique carry out house of correction acquisition can be specific binding with prostate specific membrane antigen antibody.
9. the affine adsorbing material of the immunity for prostate specific membrane antigen, including carrier, it is 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.
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