CN105968203A - Single-domain heavy chain antibody for anti-prostate specific membrane antigen extracellular region - Google Patents
Single-domain heavy chain antibody for anti-prostate specific membrane antigen extracellular region Download PDFInfo
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Abstract
The invention relates to a single-domain heavy chain antibody aiming at anti-prostate specific membrane antigen, which belongs to the field of gene engineering. The antibody has the protein with the amino acid sequence disclosed as SEQ ID NO:1, and can be used in the fields of immune detection, antigen enrichment purification and the like. The amino acid sequence provided by the invention can be used as a precursor, can be modified by using a random or site-directed mutagenesis technique to obtain a mutant with better properties (affinity, specificity, stability and the like), and is used for developing proteins or polypeptides which are further used in medicine, industry and agriculture.
Description
Technical field
The present invention relates to single domain heavy chain antibody technology (also known as nano antibody technology), and genetic engineering
Antibody technique, 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, variable domain of heavy chain
Of heavy-chain antibody) refer to only by heavy chain antibody (heavy-chain antibody) variable region
The genetic engineering antibody that (Variable region) forms, wherein, heavy chain antibody (heavy-chain
Antibody) it is a kind of to be present in the antibody of natural deletions light chain in the animal body such as camel, shark.Single
Territory heavy chain antibody is the minimum intact antigen binding fragment being currently known, and has that molecular weight is little, infiltration
Property the feature such as good, be widely used in basic research, medical diagnosis and detection, antibody drug exploitation at present
In field.
Carcinoma of prostate is the malignant tumor that a kind of serious threat elderly men is healthy, its early diagnosis and controlling
Treat and its prognosis is had great importance.Prostate specific membrane antigen (prostate specific
Membrane antigen, PSMA) it is a kind of II type transmembrane glycopeptide being positioned on prostatic cell film
In vain, it is made up of 750 aminoacid, is divided into intracellular region (aminoacid sequence is 1-18), cross-film district (19-43)
With extracellular region (44-750), relative to the prostate specific antigen (prostate being conventionally used to Clinical detection
Specific antigen, PSA), it is a kind of more sensitive and special prostate cancer label, especially
It is to be high expressed in hormone-refractory prostate cancer and carcinoma of prostate metastasis, is distinguishing prostatitis
Adenocarcinoma and the sensitivity of other types malignant tumor and specificity are respectively 65.9% and 94.5%.It addition,
Multiple non-prostate source solid tumor, as pulmonary carcinoma, bladder cancer, gastric cancer, cancer of pancreas, renal carcinoma and
Colorectal cancers etc., are 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 in addition extracellular region of 707 aminoacid compositions
The features such as multiple epitopes can be provided so that it is become tumour immunity targeted therapy and molecular image
Research target spot important in.
At present, it has been found that multiple being also prepared for for PSMA multiple can occur specificity to tie with it
The material closed, including monoclonal antibody 7E11, J591, fit A9 and A10, recombinant technique obtains
ScFv antibody D7, Fab antibody and the report of humanized antibody, passed through U.S. FDA
Approval for carcinoma of prostate diagnosis and late period radionuclide therapy 111In-pendetide, it is simply that based on for
The monoclonal antibody of PSMA is connected with radionuclide and forms.But compared with single domain heavy chain antibody, this
The shortcomings such as it is of a relatively high that a little parts exist production cost, and preparation process is complicated.
Summary of the invention
It is an object of the invention to provide the single domain heavy chain antibody for prostate specific membrane antigen, permissible
It is used for reagent and the instrument of preparation detection prostate specific membrane antigen.
The present invention provides single domain heavy chain antibody (the i.e. present invention for prostate specific membrane antigen
A kind of single domain heavy chain antibody of anti-prostate-specific membrane antigen extracellular region), there is SEQ ID NO.:1
Shown aminoacid sequence, its aminoacid sequence can pass through standardized antibody amino acids sequence numbering side
Method (ImMunoGeneTics, IMGT) is numbered the division with domain.
The present invention provides a kind of protein or polypeptide, it is characterized in that comprising in framework region one or
Plural aminoacid sequence, and at least with an aminoacid sequence, there is 90% homology.
The present invention provides a kind of protein or polypeptide, it is characterized in that comprising in complementary determining region one or
The plural aminoacid sequence of person, and at least with an aminoacid sequence, there is 80% homology.
The present invention provides a nucleic acid molecules, it is characterized in that encoding SEQ ID NO.:1, close by heredity
Numeral can obtain the particular sequence of this nucleic acid molecules at any time.The sequence of this nucleic acid molecules such as SEQ ID
NO.:2。
The present invention also provides for a nucleic acid molecules, it is characterized in that encoding SEQ ID NO.:1 part-structure
Territory, can obtain the particular sequence of this nucleic acid molecules at any time by genetic codon.
Nucleotide sequence provided by the present invention or at least partly sequence can be by suitably expressing
System carries out expressing to obtain corresponding protein or polypeptide.These expression systems include antibacterial, yeast
Bacterium, 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 letter
And property, 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.The protein provided based on the present invention or polypeptide and prostate specific
The ability that membrane antigen is specific binding, sets up the detection method of prostate specific membrane antigen.Wherein,
Preferably method include enzyme linked immunosorbent assay (Enzyme-linked immunosorbent assay,
ELISA), fluorescent immune method (Fluoroimmunoassay, FIA), immuno-chip method, affinity chromatograph
Method and immunochromatographic method.
The present invention is directed to prostate specific membrane antigen single domain heavy chain antibody at non-diseases diagnoses and treatment mesh
Immune detection and thalline or antigen enrichment purification in application.
Aminoacid sequence provided by the present invention can be as precursor, by random or site-directed mutagenesis technique
Transform, it is possible to obtain character (water solublity, stability, affinity and specificity etc.) more preferable
Mutant, be used for develop be further used for medicine, industry, agricultural protein or polypeptide.
The invention still further relates to a kind of affine adsorbing material of the immunity for prostate specific membrane antigen, bag
Including carrier, the aglucon being mounted on carrier, this material is with the single domain for prostate specific membrane antigen
Heavy chain antibody has as aglucon, the described single domain heavy chain antibody for prostate specific membrane antigen
Aminoacid sequence shown in SEQ ID NO.:1.Described carrier is magnetic bead, agarose gel microsphere, silicon
Glue microsphere or porous material.
Some terms that the present invention is described have a following implication:
Homology: describe the similarity degree of two or more aminoacid sequences, first aminoacid sequence
With the percentage ratio of homology can be by [in first aminoacid sequence between second aminoacid sequence
The number of the amino acid residue identical with the aminoacid sequence of corresponding position in second aminoacid sequence
Amount] calculate being multiplied by [100%] divided by [in first aminoacid sequence aminoacid sum],
Wherein certain the amino acid whose disappearance in second aminoacid sequence, insert, replace or add (with
One aminoacid is compared) it is considered as to have difference.Alternatively, percent homology can also utilize
The Computing program such as NCBI Blast for sequences match known obtains.
Domain: the fundamental structural unit of tertiary protein structure, is generally of certain function.
IMGT numbers: IMGT data base (The International ImMunoGeneTics Datbase)
In one the most standardized antibody amino acids sequence method for numbering serial.Concrete method for numbering serial is referred to
Document (Ehrenman, F., Q.Kaas, et al. (2010). " IMGT/3D structure-DB and
IMGT/DomainGapAlign:a databaseand a tool for immunoglobulins or
antibodies,T cell receptors,MHC,IgSF and MhcSF.”Nucleic Acids 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):55-77.
Http:// www.imgt.org/) in description.
Codon (codon): be also called three disjunctor codons (triplet code), refer to corresponding to certain
Amino acid whose nucleotide triplet.Polypeptide chain in this kind of aminoacid insertion growth is determined during translation
Position.
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 big rule of biological method can be passed through
The character such as mould production, low cost, efficient, little, the good penetrability of molecular weight, demonstrate good application
Prospect.
Accompanying drawing explanation
Fig. 1 bacterium colony PCR primer electrophoresis, recombiant protein electrophoresis and Western Blot identify figure.Left side Marker
Swimming lane is DNA molecular amount standard, and the bacterium colony PCR fragment in swimming lane 1 occurs in desired location.In
Between carry out SDS-PAGE detection for albumen after purification, in desired location, bright band occurs.The right
Figure is identified for the Western Blot with anti-PSMA monoclonal antibody and anti-His tag antibody.
Detailed description of the invention
Below by single domain heavy chain antibody (polypeptide) preparation, analyze and apply, the present invention is done into
One step explanation, these specific embodiments are not construed in any way as limiting the application model of the present invention
Enclose.
Embodiment 1
The eukaryotic expression of prostate specific membrane antigen film outskirt
RNAiso Plus reagent is used to extract total serum IgE in the LNCaP cell of high expressed PSMA,
Utilize RT-PCR method to obtain encoding the DNA fragmentation of PSMA extracellular domain fragment, use Not I
It is inserted into carrier for expression of eukaryon pRAG2a with I two kinds of restricted enzyme of BamH, passes through
T4DNA ligase is connected as recombiant plasmid.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 fat
Plastid Lipofectamine 2000 will cultivate, in not in DNA plasmid transfection to HEK-293 cell
Phase point collects supernatant simultaneously, carries out SDS-PAGE electrophoresis detection.After cultivating certain time, according to
HisTrap FF Crude test kit operation this albumen of purification.Albumen after purification is carried out SDS-PAGE
After, electricity goes on pvdf membrane, after 5% defatted milk powder is closed, is separately added into PSMA antibody and His
Antibody, 4 DEG C overnight；After rinsing, the anti-incubated at room temperature 1h that adds two, again rinse, add nitrite ion
Carry out develop (Fig. 1).
Embodiment 2
Anti-prostate specific membrane antigen single domain heavy chain antibody is (i.e. for anti-prostate specific membrane antigen single domain
Heavy chain antibody) elutriation and qualification
Use the method for solid phase elutriation from camel source natural antibody phage display library (for list of references:
" painting chases after, Xu Yang, Liu Xia, etc. the structure in camel natural single domain heavy chain antibody storehouse, source and qualification [J]. in
State's biological engineering magazine, 2011,31 (4): 31-36. " display libraries built in) in elutriation pin
Single domain heavy chain antibody to prostate specific membrane antigen.The table of prostate specific membrane antigen film outskirt
Reach and carry out according to the above embodiments 1, during first round elutriation, employing phosphate buffered solution (PBS,
PH7.4) (2-4 wheel is coated dense to 150 μ g/mL to dilute the PSMA extracellular region protein of above-mentioned synthesis
Degree is respectively 100,50,50 μ g/mL), in ELISA Plate, every hole adds 100 μ L, and 4 DEG C were coated
Night.PBST (containing 0.5%Tween 20) washes plate 5 times, and 3%BSA-PBS closes 2h at 37 DEG C,
Wash plate 3 times, add the phage antibody library 100 μ L hatched with 0.5%BSA-PBS (containing about 2
×1011CFU), hatch 1h for 37 DEG C, wash plate 5 times (increasing by 3 times by wheel) with PBST, then use PBS
Wash plate 10 times (increasing by 5 times by wheel).Again with 100 μ L eluents (glycine-HCI, pH 2.2)
The phage (37 DEG C, 5min) of eluting absorption, with 50 μ L Tris-HCl (1mol/L, pH 9.0)
Neutralize eluate, take 10 μ L for titer determination, for next round elutriation after remaining eluate amplification.
The PSMA extracellular region protein using concentration to be 5 μ g/mL after four-wheel elutriation is coated enzyme mark
Plate, washes plate, closes ibid.The phage clone 37 DEG C adding amplification purification hatches 15min, 100 μ L/
Hole, hatches 1h for 37 DEG C.The HRP-anti-M13 antibody that dilution factor is 1:5000 is added after washing plate,
100 μ L/ holes, hatch 1h for 37 DEG C.PBST washes plate 5 times, adds TMB working solution 100 μ L/ hole,
Room temperature 20min, every hole adds 50 μ L sulphuric acid (concentration is 2mol/L) and terminates reaction, measures 450
Nm light absorption value.Using previous round amplification phage antibody library direct coated ELISA Plate as positive control,
With PBS replacement phage clone as blank, measure phage by indirect phage-ELISA method
The combination activity of grain.
The indirect phage-ELISA of table 1 is loaded table
Send order-checking company to carry out sequencing ELISA positive colony, obtain anti-prostate specific membrane antigen
The Insert Fragment DNA sequence of single domain heavy chain antibody phage positive colony, is specially (SEQ ID
NO.:2):
ATGGCCCAGTTGCAGCTCGTGGAGTCCGGGGGAGGATTGGTGCAGGCTGGGGGCTCT
CTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTAGGTATGCCATGGGCTGGTT
CCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCCGCTATTAGCCGGAGTGGTGTT
AATACGCGCTATGAAGACTCCGTGAAGGGCCGATCAACCATCTCCAGAGACAACGCCA
AGAGGTCGGTGTTTCTGCAAATGGACAGTCTGAAACGTGACGACACGGCCGTTTATTA
CTGTGCAGCCCGACGTGGGCCGGGTACTTCTGTTTTGAGTGATGATTATGACTACTGGG
GTCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAGCGG
CCGC
The aminoacid sequence of its coding is as shown in SEQ ID NO.:1:
QLQLVESGGGLVQAGGSLRLSCAASGRTFSRYAMGWFRQAPGKEREFVAAISRSGVNTRY
EDSVKGRSTISRDNAKRSVFLQMDSLKRDDTAVYYCAARRGPGTSVLSDDYDYWGQGT
QVTVSS.The i.e. single domain heavy chain antibody of the anti-prostate specific membrane antigen of the present invention, its energy and PSMA
The film outskirt of albumen occurs specific binding.
Embodiment 3
The ELISA of PSMA express cell and fluorescence immunoassay detection
Gastric cancer cell MKN45 does not express PSMA, and prostate gland cancer cell LNCaP expresses PSMA,
As a example by both cells, use the anti-prostate specific membrane antigen list that in embodiment 2, elutriation obtains
Territory heavy chain antibody phage positive colony carries out ELISA and the fluorescence immunoassay detection of cellular level.To
96 orifice plates are planted respectively into LNCaP cell (expressing PSMA) and MKN45 cell (not expressing PSMA)
Each 1 × 104Individual, overnight incubation.4% paraformaldehyde is fixed, and every hole drips 3% peroxidating of 100 μ L
Hydrogen liquid, in order to block endogenous peroxidase activity, hatches 30min for 37 DEG C.TBS washes plate 3 times,
5%BSA-PBS closes, and adds 100 μ L anti-prostate specific membrane antigen single domain heavy chain antibody phagocytosis
Body positive colony, hatches 1h in 37 DEG C.Thereafter PBS rinsing, interpolation HRP-anti-M13 antibody,
TMB working solution and OD450Mensuration with embodiment 2.Positive control uses phage to substitute cell,
Blank uses PBS to substitute the phage clone added, and is repeated 3 times.
Plant into LNCaP cell and MKN45 thin after cell climbing sheet is added in every hole in 24 orifice plates
Born of the same parents each 4 × 105Individual, overnight incubation.Taking out creep plate, 4% paraformaldehyde is fixed, and rinsing drips 3%
Hydrogen peroxide is on creep plate surface, in order to block endogenous peroxidase activity.Thereafter TBS washes 3
Secondary, 5%BSA-PBS closes 30min.Drip 100 μ L and show biting of nano antibody of the present invention
Thalline clone hatch 1h, be not added with phage clone cell climbing sheet as blank.Thereafter add dilute
Degree of releasing is that the anti-M13 monoclonal antibody of 1:2000 hatches 30min.30 μ l dilutions are dripped after developing a film
The FITC-goat anti-mouse two that degree is 1:200 resists, and lucifuge hatches 30min, and uses DAPI dyeing liquor
Redye, be placed in fluorescence microscopy Microscopic observation.
Result shows: due to do not express the MKN45 cell measured value of PSMA statistically with blank
Comparison there are differences, and shows that this phage can occur non-specific binding with it；But LNCaP
Cell measured value is significantly higher than MKN45 cell, before showing that this part variation comes from PSMA and resists
Row gland specific membrane antigen single domain heavy chain antibody phage positive colony specific binding.Meanwhile, carefully
Born of the same parents' immunofluorescence shows that LNCaP cell can be with the single domain heavy chain for prostate specific membrane antigen
Antibody positive clone combine, and MKN45 cell and do not add anti-prostate specific membrane antigen single domain
The cell climbing sheet of heavy chain antibody phage positive colony is negative, shows the anti-prostate-specific that elutriation goes out
Property membrane antigen single domain heavy chain antibody phage positive colony can effectively express positive with PSMA
Cell occurs specific binding.
Embodiment 4
The expression in escherichia coli of the single domain heavy chain antibody of anti-prostate specific membrane antigen
Extract in embodiment 2 single domain heavy chain antibody positive colony for prostate specific membrane antigen
Phasmid is template, designs primer amplified genes of interest, amplification condition: 94 DEG C of 5min
Denaturation；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.With 1% agarose gel electrophoresis detection after end, and cut glue recovery purpose fragment.
To obtain encoding the single domain heavy chain antibody of anti-prostate specific membrane antigen provided by the present invention
Double digestion gene fragment clone be connected to expression vector pET-28a, after sequence verification, obtain weight
Group plasmid.
Recombinant plasmid transformed is in e. coli bl21 (Rosseta), and picking list bacterium colony lures
Lead expression.By single colony inoculation in the test tube of LB culture medium, 37 DEG C of shaken cultivation, activated overnight；
Next day, transfer in fresh LB fluid medium in the ratio of 1%, 37 DEG C, 250rpm vibration
Cultivate, to OD600After being about 0.6, add the IPTG of final concentration of 0.1mM, 37 DEG C, 250rpm
Induction 4h.
The 2mL bacterium solution of above-mentioned induction obtains thalline by 8000rpm is centrifugal, and thalline uses aseptic PBS
Washing 3 times, and carry out resuspended thalline with the aseptic PBS of 1mL, ultrasonication thalline is until bacterium on ice
Liquid is limpid, is centrifuged cell pyrolysis liquid at 4 DEG C, and centrifugal condition is 12000rmp/min, time
Between be 10min, take supernatant and add 5 μ l 5 × SDS sample-loading buffers, boiling water boils 5min, centrifugal after
Take supernatant carry out SDS-PAGE electrophoretic analysis and use nickel post that it is purified.
By optimizing abduction delivering condition (such as Host Strains, expression vector, induction time, inducing temperature
And IPTG concentration etc.), the expression of destination protein (single domain heavy chain antibody) can be improved further,
Approach is provided for preparing the single domain heavy chain antibody of anti-prostate specific membrane antigen in a large number.
Embodiment 5
Mensuration for the single domain heavy chain antibody affinity costant of prostate specific membrane antigen
Biotinylation kit is used to carry out biotinylation the single domain heavy chain antibody expressed in embodiment 4
Labelling, uses thereafter the competitive ELISA technical measurement biotinylation of standard for prostate specific
The psma protein affinity that the single domain heavy chain antibody of membrane antigen is recombinant expressed with embodiment 1.Tool
Body step is: initially with the biotinylated single domain heavy chain for prostate specific membrane antigen of 1nM
Antibody PSMA antigen with 13 kinds of variable concentrations (0.1nM～100 μMs) respectively enters in EP pipe
Row 30min is hatched；Thereafter, by 90 μ L mixed liquors add used that 3%BSA-PBST closes,
It is coated with in the ELISA Plate of psma protein, after hatching 10min, inhales and abandon reactant liquor, and use PBST
Clean；Then, add the Streptavidin that 100 μ L dilution factors are 1:2000HRP labelling, incubate
PBST is used to clean 5 times after educating 1h；The use of TMB working solution and OD450Mensuration with implement
Example 2.Nonlinear regression analysis is used to obtain OD450During maximum half, corresponding PSMA concentration,
Principle according to Ag-Ab competitive binding experiment draws biotinylated for prostate specific
The single domain heavy chain antibody affinity costant of membrane antigen is 5 × 10-7About/M.
The preparation of the immune affine adsorbing material of embodiment 6
1, the preparation of immune affine magnetic bead
Nanometer magnetic bead is used to resist for the single domain heavy chain of prostate specific membrane antigen as carrier, coupling
After body, obtain the single domain heavy chain antibody immunomagnetic beads for prostate specific membrane antigen, specifically prepare
Method is as follows:
The magnetic bead taking 1mg carboxyl modified (transports nanosecond science and technology company limited, carboxyl magnetic bead purchased from Wuxi hundred
300nm) in centrifuge tube, add 500 μ l activation buffer (10mM, NaH2PO4, pH 6.0),
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, stand 30min.
With coupling buffer (10mM, Na2HPO4, pH 7.4) and wash magnetic bead 3 times, add and be dissolved in idol
Single domain heavy chain antibody 1mg, the room temperature reaction 3h for prostate specific membrane antigen of connection buffer,
Wash magnetic bead 3 times with coupling buffer, add 500 μ l containing 1% (w/v) bovine serum albumin
(BSA) coupling buffer of or 1% (w/v) ovalbumin (OVA) closes unreacted work
Property group, room temperature reaction 30min.Magnetic bead is washed 3 times with coupling buffer, PBS solution (10mM,
PH7.4,0.02%w/v, Na3N) it is stored in 4 DEG C after resuspended.
2, for the single domain heavy chain antibody affine adsorbing material of immunity and the affinity column of prostate specific membrane antigen
Preparation.Use agarose microbeads as carrier, the single domain heavy chain of coupling prostate specific membrane antigen
Antibody, concrete preparation method is as follows:
The dry glue 0.1M HCl activated by CNBr washs 10 times, balances 5min every time.By idol
Connection buffer (10mM, Na2HPO4, pH 7.4) wash 10 times, add for prostate-specific
The single domain heavy chain antibody (2mg/ every gram agarose microbeads) of property membrane antigen, room temperature reaction 4h, make for
The agarose gel microsphere covalency that the single domain heavy chain antibody of prostate specific membrane antigen activates with CNBr
Coupling.With coupling buffer (10mM, Na2HPO4, pH 7.4) washing 2 times after, add envelope
Close liquid room temperature reaction 2h to close unreacted active group.With the long-pending phosphate buffer of 5 times of colloids
Alternately washing 3 times of (10mM, pH 7.4) and acetate buffer solution (0.1M, pH 4.0), obtain
Covalent coupling is for the immunity affine adsorption material of the single domain heavy chain antibody of prostate specific membrane antigen
Material.Taking the 0.2ml affine adsorbing material of above-mentioned immunity in capacity is the chromatographic column of 1ml, 5～10 times of posts
After PBS (10mM, the pH 7.4) washing of bed volume, add 20% ethanol solution, 4 DEG C of preservations.
3, for the single domain heavy chain antibody affine adsorbing material of immunity and the affinity column of prostate specific membrane antigen
Preparation.Use silica gel microball as carrier, the single domain heavy chain of the anti-prostate specific membrane antigen of coupling
Antibody, concrete preparation method is as follows:
Take 2g silica gel microball pure water and phosphate buffer (PBS, 10mM, pH 6.0) is alternately washed
Wash 5～10 times, with 10ml PBS suspension silica gel microball, add 5mg special for prostate
The single domain heavy chain antibody of specific membran antigene, mixing, add the carbodiimide of final concentration 5mg/ml
(EDC), mix rapidly, 4 DEG C of stirring reactions 12～24h, obtain covalent coupling for prostate
The affine adsorbing material of immunity of the single domain heavy chain antibody of specific membrane antigen.Take 0.2ml above-mentioned immunity parent
It is the chromatographic column of 1ml with adsorbing material in capacity, the PBS of 5～10 times of bed volumes (10mM,
PH 6) after washing, add containing 0.02% (w/v) Na3The PBS (10mM, pH 6) of N, 4 DEG C
Preserve.
The immunity affine adsorbing material aglucon that the present invention is directed to prostate specific membrane antigen is single domain weight
Chain antibody, has the aminoacid sequence shown in SEQ ID NO.:1, and this aglucon can specific recognition prostatitis
Gland specific membrane antigen.This single domain heavy chain antibody is readily available, adsorption efficiency high, can be by biology
It is single domain heavy chain antibody that method mass propgation produces aglucon, it is to avoid the loaded down with trivial details producers such as artificial antibody
Method, greatly reduces production cost.There is acid and alkali-resistance, high temperature resistant and the characteristic such as be readily produced, this
A little characteristics are examined for the low cost of prostate specific membrane antigen, reusable purification and immunology
Survey method has important practical value.
Claims (9)
1., for the single domain heavy chain antibody of prostate specific membrane antigen, there is SEQ ID NO.:1 institute
The aminoacid sequence shown.
2. a nucleic acid molecules, is characterized in that encoding aminoacid sequence described in claim 1.
Nucleic acid molecules the most according to claim 2, it is characterised in that sequence such as SEQ ID
NO.:2。
4. the carrier of the nucleotide sequence comprised described in claim 2.
5. the host cell of the carrier comprised described in claim 4.
6. described in claim 1 for prostate specific membrane antigen single domain heavy chain antibody in immunity
Application in detection, thalline or antigen enrichment purification.
7. the single domain heavy chain antibody for prostate specific membrane antigen described in claim 1 is in system
For the application in prostate specific membrane antigen immune detection, enrichment and purified reagent or material.
8. the single domain heavy chain antibody for prostate specific membrane antigen described in claim 1 passes through
What random or site-directed mutagenesis technique carried out house of correction acquisition can be with prostate specific membrane antigen specificity
In conjunction with antibody.
9., for the affine adsorbing material of immunity of prostate specific membrane antigen, including carrier, take
The aglucon being loaded on carrier, it is characterised in that this material is with the single domain for prostate specific membrane antigen
Heavy chain antibody has as aglucon, the described single domain heavy chain antibody for prostate specific membrane antigen
Aminoacid sequence shown in SEQ ID NO.:1.
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| CN113710707B (en) * | 2019-04-05 | 2024-05-31 | 特尼奥生物股份有限公司 | Heavy chain antibodies that bind to PSMA |
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