CN105524171A - VHH (variable domain of heavy chain of heavy-chain) antibody for prostate specific membrane antigen - Google Patents

Abstract

The invention belongs to the field of genetic engineering, specifically a single-domain heavy chain antibody for prostate-specific membrane antigen, which has SEQ? ID? The amino acid sequence shown in NO.1 can be used in the fields of immunoassay, antigen enrichment and purification, etc. The amino acid sequence provided by the present invention can be used as a precursor to be modified by random or site-directed mutation techniques to obtain mutants with better properties (affinity, specificity, stability, etc.), which can be used to develop further applications in medicine, Industrial, agricultural proteins or peptides.

Description

A Nanobody Against Prostate-Specific Membrane Antigen

technical field

The present invention relates to single domain heavy chain antibody technology (also known as nanobody technology), and genetic engineering antibody technology, especially single domain heavy chain antibody or polypeptide for prostate specific membrane antigen.

technical background

Single-domain antibodies refer to genetically engineered antibodies composed of common antibody variable regions (VH or VL). Single-domain heavy chain antibody (also known as nanobody, VHH antibody, variable domain of heavy chain of heavy-chain antibody) refers to a genetically engineered antibody composed only of heavy-chain antibody variable region (Variable region), wherein heavy-chain antibody (heavy-chain antibody) -chainantibody) is an antibody that naturally lacks light chains in animals such as camels and sharks. Single domain heavy chain antibody is the smallest complete antigen-binding fragment known so far. It has the characteristics of small molecular weight and good permeability. It has been widely used in basic research, medical diagnosis and detection, antibody drug development and other fields.

Prostate cancer is a malignant tumor that seriously threatens the health of elderly men, and its early diagnosis and treatment are of great significance to its prognosis. Prostate specific membrane antigen (PSMA) is a type II transmembrane glycoprotein located on the prostate cell membrane. It consists of 750 amino acids and is divided into intracellular region (amino acid sequence 1-18), transmembrane region (19 -43) and the extracellular region (44-750), compared with the traditional prostate specific antigen (PSA) used for clinical detection, it is a more sensitive and specific prostate cancer tumor marker, especially in hormone refractory It is highly expressed in prostate cancer and prostate cancer metastases, and its sensitivity and specificity in distinguishing prostate cancer from other types of malignant tumors are 65.9% and 94.5%, respectively. In addition, in a variety of non-prostatic solid tumors, such as lung cancer, bladder cancer, gastric cancer, pancreatic cancer, kidney cancer and colorectal cancer, PSMA is also highly specifically expressed on tumor vascular endothelial cells. Moreover, it has the activity of neurocarboxypeptidase and folate hydrolase, and the extracellular region composed of 707 amino acids can provide multiple epitopes, making it an important research in tumor immune targeting therapy and molecular imaging. target.

At present, people have discovered a variety of substances that can specifically bind to PSMA, including monoclonal antibodies 7E11 and J591, aptamers A9 and A10, ScFv antibodies D7, Fab antibodies and human-derived antibodies obtained by recombinant technology. The report of the antibody, especially 111In-pentipeptide, which has been approved by the US FDA for the diagnosis of prostate cancer and the treatment of advanced nuclide, is based on the monoclonal antibody against PSMA linked to the radionuclide. However, compared with single-domain heavy-chain antibodies, these ligands have disadvantages such as relatively high production costs and complicated preparation processes.

Contents of the invention

The purpose of the present invention is to provide a single-domain heavy chain antibody against prostate-specific membrane antigen, which can be used to prepare reagents and tools for detecting prostate-specific membrane antigen.

The present invention provides a single-domain heavy chain antibody against prostate-specific membrane antigen (that is, a nanobody against prostate-specific membrane antigen of the present invention), which has the amino acid sequence shown in SEQ ID NO.: 1, and its amino acid sequence can be standardized The antibody amino acid sequence numbering method (ImMunoGeneTics, IMGT) was used for numbering and domain division.

The present invention provides a protein or polypeptide, which is characterized by comprising one or more than two amino acid sequences in the framework region, and having at least 90% homology with one amino acid sequence.

The present invention provides a protein or polypeptide, which is characterized by comprising one or more than two amino acid sequences in the complementarity determining region, and having at least 80% homology with one amino acid sequence.

The present invention provides a nucleic acid molecule, which is characterized by encoding SEQ ID NO.: 1, and the specific sequence of the nucleic acid molecule can be obtained at any time through the genetic code. The sequence of the nucleic acid molecule is shown as SEQ ID NO.:2.

The present invention also provides a nucleic acid molecule, which is characterized in that it encodes a partial structural domain of SEQ ID NO.: 1, and the specific sequence of the nucleic acid molecule can be obtained at any time through the genetic code.

The nucleotide sequence or at least a part of the sequence provided by the present invention can be expressed through a suitable expression system to obtain the corresponding protein or polypeptide. These expression systems include bacteria, yeast, filamentous fungi, animal cells, insect cells, plant cells, or cell-free expression systems.

The present invention also provides a vector comprising the nucleic acid sequence. Due to the degeneracy of the genetic code, the nucleic acid sequence may vary according to different application purposes.

The present invention also provides a host cell including the protein or expression vector.

The present invention also provides a method for detecting prostate-specific membrane antigen on cells, which is characterized by containing the above-mentioned protein or polypeptide. Based on the ability of the protein or polypeptide provided by the invention to specifically bind to the prostate-specific membrane antigen, a detection method for the prostate-specific membrane antigen is established. Among them, preferred methods include enzyme-linked immunosorbent assay (Enzyme-linkedimmunosorbentassay, ELISA), fluorescence immunoassay (Fluoroimmunoassay, FIA), immunochip method, affinity chromatography and immunochromatography.

The invention is aimed at the application of the prostate-specific membrane antigen single-domain heavy chain antibody in immunodetection for non-disease diagnosis and treatment purposes, and in the enrichment and purification of bacteria or antigens.

The amino acid sequence provided by the present invention can be used as a precursor to be modified by random or site-directed mutation techniques to obtain mutants with better properties (water solubility, stability, affinity and specificity, etc.) for development and further use in medicine , industrial, agricultural proteins or peptides.

The present invention also relates to an immunoaffinity adsorption material for prostate-specific membrane antigen, including a carrier and a ligand carried on the carrier. The material uses a single-domain heavy chain antibody for prostate-specific membrane antigen as a ligand, so The single domain heavy chain antibody against prostate specific membrane antigen has the amino acid sequence shown in SEQ ID NO.:1. The carrier is magnetic beads, agarose gel microspheres, silica gel microspheres or porous materials.

Some terms described in the present invention have the following meanings:

Homology: Describes the degree of similarity between two or more amino acid sequences. The percentage of homology between the first amino acid sequence and the second amino acid sequence can be determined by [In the first amino acid sequence and in the second amino acid sequence The number of amino acid residues with the same amino acid sequence at the corresponding position] divided by [the total number of amino acids in the first amino acid sequence] multiplied by [100%] to calculate, wherein the deletion of a certain amino acid in the second amino acid sequence, Insertions, substitutions or additions (compared to the first amino acid) are considered to be different. Alternatively, percent homology can also be obtained using known computer algorithms for sequence matching such as NCBI Blast.

Domain: The basic structural unit of the tertiary structure of a protein, usually with a certain function.

IMGT numbering: A standardized antibody amino acid sequence numbering method in the IMGT database (TheInternationalImMunoGeneTicsDatbase). The specific numbering method can refer to the literature (Ehrenman, F., Q.Kaas, et al. (2010). "IMGT/3Dstructure-DBandIMGT/DomainGapAlign: adatabaseandatoolforimmunoglobulinsorantibodies, Tcellreceptors, MHC, IgSFandMhcSF."NucleicAcidsRes38(Databaseissue): D301-307.Lef , M.P., C.Pommie, et al. (2003). "IMGTuniquenumberingforimmunoglobulinandTcellreceptorvariabledomainsandIgsuperfamilyV-likedomains"DevcompImmunol27(1): 55-77. http://www.imgt.org/).

Codon: also known as triplet codon (tripletcode), refers to a nucleotide triplet corresponding to a certain amino acid. The insertion of this amino acid into the growing polypeptide chain is determined during translation.

Beneficial effects of the present invention: the single-domain heavy chain antibody or polypeptide directed against prostate-specific membrane antigen of the present invention has specific binding to prostate-specific membrane antigen, can be mass-produced by biological methods, has low cost, high efficiency, small molecular weight, Good permeability and other properties, showing good application prospects.

Description of drawings

Fig. 1 Colony PCR product electrophoresis, recombinant protein electrophoresis and Western Blot identification diagram. The marker lane on the left is the DNA molecular weight standard, and the colony PCR fragment in lane 1 appears at the expected position. In the middle is the purified protein for SDS-PAGE detection, and a bright band appears at the expected position. On the right is the WesternBlot identification chart with anti-PSMA monoclonal antibody and anti-His tag antibody.

detailed description

The present invention will be further described through the preparation, analysis and application of single domain heavy chain antibody (polypeptide) below, and these specific examples should not be interpreted as limiting the scope of application of the present invention in any way.

Example 1

Eukaryotic Expression of the Extramembrane Region of Prostate Specific Membrane Antigen

RNAisoPlus reagent was used to extract the total RNA in LNCaP cells with high expression of PSMA, and the DNA fragment encoding the extracellular region of PSMA was obtained by RT-PCR method, which was inserted into the eukaryotic expression vector pRAG2a using NotI and BamHI two restriction enzymes, Ligated into a recombinant plasmid by T4 DNA ligase. The recombinant plasmid was heat-shocked and transformed into TOP10 competent cells and cultured overnight, and the correctly identified clones were sent for sequencing verification. The plasmids of positive clones were extracted, and the DNA plasmids were transfected into HEK-293 cells using liposome Lipofectamine2000 for culture, and the supernatants were collected at different time points for SDS-PAGE electrophoresis detection. After culturing for a certain period of time, the protein was purified according to the operation of the HisTrapFFCrude kit. After the purified protein was subjected to SDS-PAGE, it was electrotransferred to PVDF membrane, and after blocking with 5% skimmed milk powder, PSMA antibody and His antibody were added respectively, overnight at 4°C; after rinsing, the secondary antibody was added to incubate at room temperature for 1 h, and then rinsed again. Add chromogenic solution for development (Figure 1).

Example 2

Panning and Identification of Anti-Prostate-Specific Membrane Antigen Single-Domain Heavy-Chain Antibody

Using solid-phase panning method to display camel-derived natural antibody phage display library (for reference: "Tu Zhui, Xu Yang, Liu Xia, et al. Construction and identification of camel-derived natural single-domain heavy chain antibody library [J]. China Bioengineering Journal, 2011, 31 (4): 31-36." In the display library constructed in "), the single-domain heavy chain antibody against prostate-specific membrane antigen was panned. The expression of the extracellular region of prostate-specific membrane antigen was carried out according to the above-mentioned Example 1. During the first round of panning, the protein of the extracellular region of PSMA synthesized above was diluted to 150 g/mL with phosphate buffered saline solution (PBS, pH7.4) (The coating concentrations of the second to fourth rounds are 100, 50, and 50 g/mL, respectively), add 100 μL to each well of the microtiter plate, and coat overnight at 4°C. Wash the plate 5 times with PBST (containing 0.5% Tween20), block with 3% BSA-PBS at 37°C for 2 hours, wash the plate 3 times, add 100 μL of phage antibody library (containing about 2×10 ¹¹ CFU) incubated with 0.5% BSA-PBS ), incubated at 37°C for 1 h, washed the plate 5 times with PBST (increase 3 times for each round), and then washed the plate 10 times with PBS (increased 5 times for each round). The adsorbed phages were then eluted with 100 μL eluent (glycine-hydrochloric acid, pH 2.2) (37°C, 5 min), neutralized with 50 μL Tris-HCl (1mol/L, pH 9.0), and 10 μL was used for The titer was determined, and the remaining eluates were amplified and used for the next round of panning.

After four rounds of panning, the PSMA extracellular region protein with a concentration of 5 μg/mL was used to coat the microplate, and the plate was washed and blocked as above. Add the amplified and purified phage clone and incubate at 37°C for 15min, 100μL/well, and incubate at 37°C for 1h. After washing the plate, add HRP-anti-M13 antibody at a dilution of 1:5000, 100 μL/well, and incubate at 37° C. for 1 h. The plate was washed 5 times with PBST, 100 μL/well of TMB working solution was added, room temperature was 20 min, 50 μL of sulfuric acid (concentration: 2 mol/L) was added to each well to terminate the reaction, and the absorbance at 450 nm was measured. The phage antibody library amplified in the previous round was directly coated on the microtiter plate as a positive control, and PBS was used instead of phage clone as a blank control, and the binding activity of phage particles was determined by indirect phage-ELISA method.

Table 1 Indirect phage-ELISA loading table

The ELISA positive clone was sent to a sequencing company for sequence determination, and the DNA sequence of the insert fragment of the anti-prostate specific membrane antigen single domain heavy chain antibody phage positive clone was obtained, specifically (SEQ ID NO.: 2):

ATGGCCCAGTTGCAGCTCGTGGAGTCCGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGTAGTCTCTGGACGCCCATTCAGTAGATATACCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGCGAGTTGGTCGCAACAATTTGGCGTCGAGGTAACACATACTACGCAAACTACGCAGACGCTGTGACGGGCCATTCACCATCTCCAGAGACAACGCCAAGAACACGGCGTATCTGCAAATGAACAGCCTTGAACTTGAGGACACGGCCATTTATTACTGTGCAGCAGGCCGGACGAGTTGGGGTCAAAACCCATCGGAAIATGGCTACTGGGGCCAGGGCACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAGCGGCCGC

The encoded amino acid sequence is shown in SEQ ID NO.: 1:

QLQLVESGGGLVQAGGSLRLSCVVSGRPFSRYTMGWFRQAPGKERELVATIWRRGNTYYANYADAVTGRFTISRDNAKNTAYLQMNSLELEDTAIYYCAAGRTSWGQNPSEYGYWGQGTQVTVSS. That is, the anti-prostate specific membrane antigen single-domain heavy chain antibody of the present invention can specifically bind to the extramembrane region of PSMA protein.

Example 3

ELISA and fluorescent immunoassay of PSMA expressing cells

Gastric cancer cell MKN45 does not express PSMA, while prostate cancer cell LNCaP expresses PSMA. Taking these two types of cells as examples, the anti-prostate specific membrane antigen single-domain heavy chain antibody phage-positive clones obtained by panning in Example 2 were used for cell-level detection. ELISA and fluorescent immunoassay. 1×10 ⁴ LNCaP cells (expressing PSMA) and MKN45 cells (not expressing PSMA) were seeded into 96-well plates, and cultured overnight. After fixing with 4% paraformaldehyde, 100 μL of 3% hydrogen peroxide solution was added dropwise to each well to block endogenous peroxidase activity, and incubated at 37°C for 30 min. Wash the plate three times with TBS, block with 5% BSA-PBS, add 100 μL anti-prostate specific membrane antigen single domain heavy chain antibody phage-positive clone, and incubate at 37°C for 1 hour. Then rinse with PBS, add HRP-anti-M13 antibody, TMB working solution and measure OD ₄₅₀ as in Example 2. The positive control used phage to replace the cells, and the blank control used PBS to replace the added phage clones, and repeated 3 times.

After adding cell slides to each well of the 24-well plate, 4×10 ⁵ LNCaP cells and MKN45 cells were planted and cultured overnight. The slides were taken out, fixed with 4% paraformaldehyde, rinsed, and 3% hydrogen peroxide was added dropwise on the surface of the slides to block the activity of endogenous peroxidase. Thereafter, the cells were washed 3 times with TBS and blocked with 5% BSA-PBS for 30 min. 100 μL of the phage clone displaying the Nanobody of the present invention was added dropwise and incubated for 1 h, and the cell slide without the phage clone was used as a blank control. Then add anti-M13 monoclonal antibody at a dilution of 1:2000 and incubate for 30 min. After washing, 30 μl of FITC-goat anti-mouse secondary antibody at a dilution of 1:200 was added dropwise, incubated in the dark for 30 min, counterstained with DAPI staining solution, and observed under a fluorescent microscope.

The results showed that the measured value of MKN45 cells not expressing PSMA was statistically different from that of the blank control, indicating that the phage could bind to it non-specifically; however, the measured value of LNCaP cells was significantly higher than that of MKN45 cells, indicating that this part of the difference came from PSMA Specific binding of anti-prostate-specific membrane antigen single-domain heavy-chain antibody phage-positive clones. At the same time, cell immunofluorescence showed that LNCaP cells could bind to the positive clones of the single domain heavy chain antibody against prostate specific membrane antigen, while MKN45 cells and the cell slides of the phage positive clones without anti prostate specific membrane antigen single domain heavy chain antibody It was negative, indicating that the anti-PSMA single-domain heavy-chain antibody phage-positive clones selected by panning could effectively specifically bind to PSMA-positive cells.

Example 4

Expression of single domain heavy chain antibody against prostate specific membrane antigen in Escherichia coli

Extract the phagemid of the single-domain heavy chain antibody positive clone against prostate-specific membrane antigen in Example 2 as a template, design specific primers to amplify the target gene, and the amplification conditions: 94°C 5min pre-denaturation; 94°C 30s, 55°C 30 s at ℃, 40 s at 72 ℃, a total of 30 cycles; finally, extend at 72 ℃ for 5 min. After the end, use 1% agarose gel electrophoresis to detect, and cut the gel to recover the target fragment.

Cloning the obtained double-digested gene fragment encoding the single-domain heavy chain antibody against prostate-specific membrane antigen provided by the present invention was connected to the expression vector pET-28a, and after sequencing verification, a recombinant plasmid was obtained.

The recombinant plasmid was transformed into Escherichia coli BL21 (Rosseta), and a single colony was picked for induced expression. Inoculate a single colony in a test tube of LB medium, culture with shaking at 37°C, and activate overnight; the next day, transfer it to fresh LB liquid medium at a ratio of 1%, and culture with shaking at 37°C and 250rpm until the OD ₆₀₀ is about After reaching 0.6, add IPTG with a final concentration of 0.1 mM, and induce for 4 hours at 37°C and 250 rpm.

The above induced 2mL bacterial solution was centrifuged at 8000rpm to obtain the bacterial cells, the bacterial cells were washed 3 times with sterile PBS, and the bacterial cells were resuspended with 1mL sterile PBS, the bacterial cells were ultrasonically disrupted on ice until the bacterial liquid was clear, and the bacterial cells were placed at 4°C. The cell lysate was centrifuged at 12000rmp/min for 10min, the supernatant was added to 5μl 5×SDS sample buffer, boiled in boiling water for 5min, after centrifugation, the supernatant was analyzed by SDS-PAGE electrophoresis and analyzed by nickel column It is purified.

By optimizing the induced expression conditions (such as host bacteria, expression vectors, induction time, induction temperature, and IPTG concentration, etc.), the expression level of the target protein (single domain heavy chain antibody) can be further increased, and it is possible to prepare a large amount of anti-prostate specific membrane antigen. Single domain heavy chain antibodies provide an approach.

Example 5

Determination of Affinity Constants for Single Domain Heavy Chain Antibodies Against Prostate-Specific Membrane Antigen

The single-domain heavy-chain antibody expressed in Example 4 was biotinylated using a biotinylation kit, and then a standard competitive ELISA technique was used to determine the ratio of the biotinylated single-domain heavy-chain antibody against prostate-specific membrane antigen to Affinity of recombinantly expressed PSMA protein in Example 1. The specific steps are as follows: first, 1 nM biotinylated single-domain heavy chain antibody against prostate-specific membrane antigen was used to incubate 13 kinds of PSMA antigens with different concentrations (0.1 nM-100 μM) in EP tubes for 30 min; Add 90 μL of the mixed solution to the enzyme plate coated with PSMA protein that has been blocked with 3% BSA-PBST, and after incubation for 10 min, discard the reaction solution and wash it with PBST; then, add 100 μL of HRP-labeled protein with a dilution of 1:2000 Streptavidin, washed 5 times with PBST after incubation for 1 h; the use of TMB working solution and the determination of OD ₄₅₀ were the same as in Example 2. Using nonlinear regression analysis to obtain half of the maximum value of OD ₄₅₀ , the corresponding PSMA concentration, according to the principle of antigen-antibody competitive binding experiment, the biotinylated single domain heavy chain antibody affinity constant for prostate specific membrane antigen is obtained as About 5×10 ^-7 /M.

Embodiment 6 Preparation of immunoaffinity adsorption material

1. Preparation of immunoaffinity magnetic beads

Using nano-magnetic beads as a carrier, after coupling the single-domain heavy-chain antibody against prostate-specific membrane antigen, the single-domain heavy-chain antibody immune magnetic beads for prostate-specific membrane antigen are obtained. The specific preparation method is as follows:

Take 1 mg of carboxy-modified magnetic beads (purchased from Wuxi Baiyun Nano Technology Co., Ltd., carboxy magnetic beads 300 nm) into a centrifuge tube, add 500 μl of activation buffer (10 mM, NaH ₂ PO ₄ , pH 6.0), and vortex to mix evenly , recover the magnetic beads with a magnetic stand, and wash twice with activation buffer. Add 2 mg of carbodiimide (EDC) and N-hydroxysuccinimide (NHS) respectively, vortex and mix, and let stand for 30 min. Wash the magnetic beads 3 times with coupling buffer (10mM, Na ₂ HPO ₄ , pH7.4), add 1 mg of single domain heavy chain antibody against prostate-specific membrane antigen dissolved in coupling buffer, react at room temperature for 3 h, and use Wash the magnetic beads 3 times with coupling buffer, add 500 μl of coupling buffer containing 1% (w/v) bovine serum albumin (BSA) or 1% (w/v) ovalbumin (OVA) to block unreacted Active groups, react at room temperature for 30 minutes. The magnetic beads were washed 3 times with coupling buffer, resuspended in PBS solution (10 mM, pH7.4, 0.02% w/v, Na ₃ N) and stored at 4°C.

2. Preparation of single domain heavy chain antibody immunoaffinity adsorption material and affinity column for prostate specific membrane antigen. Using agarose microspheres as a carrier, the single domain heavy chain antibody coupled to prostate specific membrane antigen, the specific preparation method is as follows:

The CNBr-activated dry gel was washed 10 times with 0.1M HCl, equilibrating for 5 min each time. Wash 10 times with coupling buffer (10mM, Na ₂ HPO ₄ , pH 7.4), add single domain heavy chain antibody against prostate specific membrane antigen (2mg/gram of agarose microspheres), react at room temperature for 4h, let A single-domain heavy-chain antibody against prostate-specific membrane antigen was covalently conjugated to CNBr-activated sepharose microspheres. After washing twice with coupling buffer (10 mM, Na ₂ HPO ₄ , pH 7.4), a blocking solution was added to react at room temperature for 2 h to block unreacted active groups. Alternately wash 3 times with phosphate buffer (10mM, pH 7.4) and acetate buffer (0.1M, pH 4.0) with 5 gel volumes to obtain a single-domain heavy chain covalently coupled to prostate-specific membrane antigen Immunoaffinity adsorption material for antibodies. Take 0.2ml of the above immunoaffinity adsorption material on a chromatographic column with a capacity of 1ml, wash with PBS (10mM, pH7.4) 5-10 times the column bed volume, add 20% ethanol solution, and store at 4°C.

3. Preparation of single domain heavy chain antibody immunoaffinity adsorption material and affinity column for prostate specific membrane antigen. Using silica gel microspheres as a carrier, coupled with a single domain heavy chain antibody against prostate specific membrane antigen, the specific preparation method is as follows:

Take 2g of silica gel microspheres and wash them alternately with pure water and phosphate buffer (PBS, 10mM, pH6.0) for 5 to 10 times, suspend the silica gel microspheres with 10ml of PBS buffer, and add 5mg of single-domain heavy chain against prostate-specific membrane antigen Antibody, mix well, add carbodiimide (EDC) with a final concentration of 5mg/ml, mix quickly, stir and react at 4°C for 12-24h, and obtain a single-domain heavy chain antibody covalently coupled to prostate-specific membrane antigen immunoaffinity adsorption material. Take 0.2ml of the above-mentioned immunoaffinity adsorption material on a chromatographic column with a capacity of 1ml, wash with PBS (10mM, pH ₆ ) of 5 to 10 times the column bed volume, and then add PBS ( 10mM, pH6), stored at 4°C.

The ligand of the immunoaffinity adsorption material for the prostate-specific membrane antigen is a single-domain heavy chain antibody, which has the amino acid sequence shown in SEQ ID NO.: 1, and the ligand can specifically recognize the prostate-specific membrane antigen. The single-domain heavy-chain antibody is easy to obtain and has high adsorption efficiency. The ligand can be mass-cultured and produced as a single-domain heavy-chain antibody by biological methods, avoiding cumbersome production methods such as artificial antibodies, and greatly reducing production costs. It has the characteristics of acid and alkali resistance, high temperature resistance and easy production, and these characteristics have important practical value for the low-cost, reusable purification and immunological detection method of prostate specific membrane antigen.

Claims (9)

1. A single-domain heavy chain antibody against prostate-specific membrane antigen, having the amino acid sequence shown in SEQ ID NO.:1. 2. A nucleic acid molecule, characterized in that it encodes the amino acid sequence described in claim 1. 3. The nucleic acid molecule according to claim 2, characterized in that the sequence is as SEQ ID NO.:2. 4. A vector comprising the nucleic acid sequence of claim 2. 5. A host cell comprising the vector of claim 4. 6. The application of the single-domain heavy chain antibody against prostate-specific membrane antigen according to claim 1 in immunoassay, bacterium or antigen enrichment and purification. 7. The application of the single-domain heavy chain antibody against prostate-specific membrane antigen of claim 1 in the preparation of reagents or materials for immunodetection, enrichment and purification of prostate-specific membrane antigen. 8. The single-domain heavy chain antibody against prostate-specific membrane antigen according to claim 1 is modified by random or site-directed mutagenesis, and is capable of specifically binding to prostate-specific membrane antigen. 9. An immunoaffinity adsorption material for prostate-specific membrane antigen, comprising a carrier, and a ligand carried on the carrier, characterized in that the material uses a single-domain heavy chain antibody for prostate-specific membrane antigen as a ligand, The single domain heavy chain antibody against prostate specific membrane antigen has the amino acid sequence shown in SEQ ID NO.:1.