Single-domain antibody aiming at NUPR1 as well as screening method and application of single-domain antibody

Abstract

The invention discloses a single-domain antibody aiming at NUPR1 as well as a screening method and application of the single-domain antibody. After the single-domain antibody is fused with a C-degron degradation element cOdc1, targeted degradation of the NUPR1 can be realized, and the progression of tumors can be effectively inhibited; after the TAT transmembrane peptide is fused, the single-domain antibody can easily enter cells, and the immunogenicity is reduced to the maximum extent.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a single-domain antibody against nuclear protein 1 (NUPR1), a screening method thereof, and an application thereof. Background Art

[0002] Nuclear protein 1 (NUPR1), also known as p8 or Com1 protein, is a nuclear-intrinsic disordered protein and stress protein. NUPR1 is transcriptionally activated under stress conditions to protect cells from stress damage. Studies have shown that NUPR1 is significantly upregulated in tumor tissues such as pancreatic cancer, breast cancer, lung cancer, and colon cancer, and participates in tumor cell migration, invasion, etc. by regulating cell activities such as autophagy, cell cycle, apoptosis, and DNA damage. Silencing NUPR1 or inhibiting its expression with an antibody may become a new method for potential cancer treatment. In view of the limitations of traditional monoclonal antibodies, such as low stability, complex preparation process, and high cost, there is an urgent need to develop new antibodies against NUPR1 currently.

[0003] A single-domain antibody (sdAb, also known as a nanobody) is a variable domain of the heavy chain (VHH) of a heavy-chain-only antibody (HcAbs) that naturally lacks a light chain and exists in the serum of camelids, with a molecular weight of about 15 kD. The sdAb has a stable structure, high affinity and antigen-binding ability, a small relative molecular mass, is easy to penetrate tissue barriers, and is easy to produce and prepare and genetically engineered. The sdAb has been widely studied and concerned due to its unique advantages, and its practice in clinical diagnosis and treatment is also actively being explored.

[0004] The traditional method for preparing sdAb uses an antigen to immunize alpacas, isolates the plasma cells of alpacas, prepares hybridoma cells, and screens out hybrid cells that can produce sdAb specifically recognizing the antigen for subsequent separation, purification, and function verification of sdAb. The whole process takes a long time, uses a large number of experimental materials, has a high cost, and the success rate of obtaining a high-affinity sdAb is extremely low. In addition, there are also some reports on the research of engineered sdAb based on the amino acid sequence of the known antibody antigen determinant region, but these studies are further supplements to the functions of existing antibodies; while the attempt to find new sdAb with higher specificity from scratch still faces great challenges. Summary of the Invention

[0005] To solve the problems existing in the prior art, the purpose of the present invention is to provide a single-domain antibody against NUPR1 and its screening method, and also provide a gene encoding the single-domain antibody, an expression vector, a host cell and related applications.

[0006] On the one hand, the present invention provides a single-domain antibody against NUPR1, which comprises three complementary determining regions, namely HCDR1, HCDR2 and HCDR3. Among them, the amino acid sequences of HCDR1, HCDR2 and HCDR3 have at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequences shown in SEQ ID NO.1 to SEQ ID NO.3 respectively. Preferably, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are respectively as shown in SEQ ID NO.1 to SEQ ID NO.3.

[0007] Furthermore, the single-domain antibody further comprises four framework regions alternately connected with the three complementary determining regions, namely FR1, FR2, FR3 and FR4. Among them, the amino acid sequences of FR1, FR2, FR3 and FR4 have at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequences shown in SEQ ID NO.4 to SEQ ID NO.7 respectively. Preferably, the amino acid sequences of FR1, FR2, FR3 and FR4 are respectively as shown in SEQ ID NO.4 to SEQ ID NO.7.

[0008] Preferably, the amino acid sequence of the single-domain antibody against NUPR1 has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO.8. Preferably, the amino acid sequence of the single-domain antibody against NUPR1 is as shown in SEQ ID NO.8, and for the convenience of description in the following text of this application, the amino acid sequence is also referred to by the code #07.81.

[0009] In another aspect, the present invention provides a fusion protein, which comprises the single-domain antibody against NUPR1. According to the present invention, the single-domain antibody against NUPR1 is as defined above.

[0010] In some embodiments of the present invention, the fusion protein further comprises the degradation element cOdc1; preferably, the fusion protein further comprises a cell-penetrating peptide.

[0011] In one embodiment of the present invention, the fusion protein comprises A-B-C-D-E-F, wherein A is a secretion peptide or absent, B is the single-domain antibody against NUPR1, C is a first tag sequence or absent, D is the degradation element cOdc1 or absent, E is a cell-penetrating peptide or absent, F is a second tag sequence or absent, and there is or is not a flexible linker between A, B, C, D, E, and / or F.

[0012] According to the present invention, the amino acid sequence of the degradation element cOdc1 is: SHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV.

[0013] According to the present invention, the cell-penetrating peptide can be an amino acid sequence known in the art to have the function of penetrating cell membranes, including but not limited to: TAT-derived peptides, antennapedia leader peptides, antennapedia peptides, polyarginine peptides, etc. For example, the amino acid sequence of the cell-penetrating peptide is YGRKKRRQRRR, GRKKRRQRRR, KKWKMRRNQFWVKVQRG, RQIKIWFQNRRMKWKK, or a polyarginine peptide containing 6-9 arginines (e.g., RRRRRR, or RRRRRRRRR). In a specific embodiment of the present invention, the amino acid sequence of the cell-penetrating peptide is YGRKKRRQRRR.

[0014] According to the present invention, the first tag sequence and the second tag sequence can be the same or different, and are selected from the tag sequences commonly used in the art, including but not limited to: His tag, Myc tag, HA tag, GST tag, mCherry, GFP, Flag tag, etc. In a specific embodiment of the present invention, the first tag sequence and the second tag sequence are different, and are respectively a His tag and a Flag tag. For example: the first tag sequence is HHHHHH, and the second tag sequence is DYKDHDGDYKDHDIDYKDDDDK; or, the first tag sequence is DYKDHDGDYKDHDIDYKDDDDK, and the second tag sequence is HHHHHH.

[0015] According to the present invention, the flexible linker can be an amino acid sequence known in the art to connect two peptide fragments, such as (Gm S) n Linker sequences, DDK linker sequences, etc. In a specific embodiment of the present invention, the sequence of the flexible linker is GSG.

[0016] Preferably, the fusion protein is A-B-C-D-E-F, wherein A is a secretion peptide or absent, B is the single-domain antibody against NUPR1, C is a first tag sequence or absent, D is the degradation element cOdc1, E is a cell-penetrating peptide, F is a second tag sequence or absent, and there is or is not a flexible linker between A, B, C, D, E, and / or F. Preferably, the amino acid sequence of the single-domain antibody against NUPR1 is as shown in SEQ ID NO.8, and / or, the amino acid sequence of the degradation element cOdc1 is SHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV, and / or, the amino acid sequence of the cell-penetrating peptide is YGRKKRRQRRR, and / or, the first tag sequence and the second tag sequence are different and are selected from HHHHHH and DYKDHDGDYKDHDIDYKDDDDK. In a specific embodiment of the present invention, the amino acid sequence of the fusion protein is MGQVQLVESGGGSVQAGGSLRLSCTASGGSEYSYSTFSLGWFRQAPGQEREAVAAIASMGGLTYYADSVKGRFTISRDNAKNTVTLQMNNLKPEDTAIYYCAALSGPSWDWECLSAIVASGELNWGQGTQVTVSSGSGDYKDHDGDYKDHDIDYKDDDDKGSGSHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINVYGRKKRRQRRRHHHHHH.

[0017] Preferably, the fusion protein is A-B-C-D-E-F, wherein A is a secretion peptide or absent, B is the single-domain antibody against NUPR1, C is a first tag sequence, D is absent, E is a cell-penetrating peptide or absent, F is absent, and there is or is not a flexible linker between A, B, C, and / or E. Preferably, the amino acid sequence of the single-domain antibody against NUPR1 is as shown in SEQ ID NO.8, and / or, the amino acid sequence of the cell-penetrating peptide is YGRKKRRQRRR, and / or, the first tag sequence is DYKDHDGDYKDHDIDYKDDDDK.

[0018] Preferably, the fusion protein is A-B-C-D-E-F, wherein A is a secretion peptide or absent, B is the single-domain antibody against NUPR1, C is absent, D is absent, E is a cell-penetrating peptide or absent, F is a second tag sequence, and there is or is not a flexible linker between A, B, E and / or F. Preferably, the amino acid sequence of the single-domain antibody against NUPR1 is as shown in SEQ ID NO.8, and / or the amino acid sequence of the cell-penetrating peptide is YGRKKRRQRRR, and / or the second tag sequence is selected from HHHHHH and DYKDHDGDYKDHDIDYKDDDDK.

[0019] In another aspect, the present invention provides an antibody preparation, which comprises the single-domain antibody against NUPR1 or the fusion protein. Further, the antibody preparation further comprises a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier includes but is not limited to: buffer solution, sterile water, surfactant, etc.

[0020] In another aspect, the present invention provides a kit, which comprises the single-domain antibody against NUPR1 or the fusion protein. The single-domain antibody against NUPR1 or the fusion protein can quantitatively or qualitatively detect whether the tested sample contains NUPR1 protein through an antigen-antibody binding reaction, so that the kit can be used to detect whether the tested sample contains NUPR1 protein.

[0021] As is known to those skilled in the art, the methods capable of detecting antigen-antibody binding reactions include but are not limited to: enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, sandwich method, immunoblotting, immunoprecipitation, immunohistochemical staining, fluorescence immunoassay, enzyme substrate coloring method, antigen-antibody aggregation method. Therefore, according to the method adopted, other chemical substances can be further included in the kit.

[0022] For example, when the method for quantitatively or qualitatively detecting through an antigen-antibody binding reaction is ELISA, the single-domain antibody against NUPR1 of the present invention included in the kit can be used as the first antibody. The first antibody is adsorbed on the surface of the solid-phase carrier. The solid-phase carrier can be selected from the solid-phase carrier materials commonly used in the art, including but not limited to: polyvinyl chloride, polystyrene, polyacrylamide or cellulose, etc. The forms of the solid-phase carrier include but are not limited to: porous plates, test tubes, beads, etc. The kit can further include a second antibody conjugated with an indicator molecule, and the second antibody can specifically bind to the NUPR1 protein, and the antigenic determinants bound by the second antibody and the first antibody are different. When the indicator molecule is an enzyme, the kit can further include a substrate of the enzyme.

[0023] For another example, the method for quantitative or qualitative detection through antigen-antibody binding reaction is immunoblotting, and the monoclonal domain antibody against NUPR1 of the present invention contained in the kit can be used as the primary antibody. The primary antibody can further bind to an indicator molecule. When the primary antibody does not bind to the indicator molecule, the kit can further contain a secondary antibody bound to the indicator molecule, and the secondary antibody can bind to the primary antibody; alternatively, the kit can further contain an IgG binding protein bound to the indicator molecule, such as protein A. The kit can also further contain reagents for electrophoretic separation of proteins.

[0024] According to the present invention, the indicator molecule can be, for example: fluorescent substances, radioactive substances, and / or enzymes, etc. The enzyme can be selected from the enzymes commonly used in the art, including but not limited to: peroxidase (such as horseradish peroxidase), alkaline phosphatase, glucose oxidase, etc. Cross-linking methods known in the art can be used to label the enzyme to the antibody, including but not limited to: glutaraldehyde method and periodate oxidation method, etc. The fluorescent substances can be selected from various fluorescent substances known and commonly used in the art, including but not limited to: fluorescein isothiocyanate, tetramethyl rhodamine, tetramethyl isothiocyanate rhodamine, phycoerythrin, Cy3, Cy5, DyLight405, DyLight 488, DyLight 550, DyLight594, DyLight 633, DyLight650, DyLight680, DyLight755, DyLight800, SYBR Green I, etc.

[0025] The sample to be detected can be a substance from a living or previously living organism, including but not limited to: blood (such as whole blood), plasma, serum, urine, amniotic fluid, synovial fluid; cells such as endothelial cells, white blood cells, monocytes; organs (heart, kidney, spleen, lung, etc.); tissues (bone marrow, lymph nodes, connective tissue, adipose tissue, etc.).

[0026] On the other hand, the present invention provides an isolated nucleic acid molecule encoding the monoclonal domain antibody against NUPR1 or the fusion protein.

[0027] Preferably, the nucleic acid molecule contains the nucleotide sequence shown in SEQ ID NO.12 or SEQ ID NO.18.

[0028] On the other hand, the present invention provides an expression vector containing the aforementioned nucleic acid molecule.

[0029] Preferably, the expression vector can be a plasmid, phage, or virus.

[0030] In another aspect, the present invention provides a host cell, wherein the genome of the host cell integrates the aforementioned nucleic acid molecule; or, the host cell contains the aforementioned expression vector. Preferably, the host cell is a eukaryotic cell or a prokaryotic cell.

[0031] In another aspect, the present invention provides a pharmaceutical composition, which contains the single-domain antibody against NUPR1 or the fusion protein. Preferably, the pharmaceutical composition further contains at least one pharmaceutically acceptable excipient. "Pharmaceutically acceptable excipients" include, but are not limited to, any and all solvents, dispersion media or other liquid carriers, dispersion or suspension aids, diluents, isotonic agents, preservatives, coloring agents, sweetening or flavoring agents, stabilizers, antioxidants, antimicrobial or antifungal agents, osmolarity regulators, pH regulators, buffers, chelating agents, cryoprotectants and / or fillers, such as those suitable for the specific dosage form required. Various excipients for formulating pharmaceutical compositions and techniques for preparing the compositions are known in the art. Exemplary antimicrobial or antifungal agents include, but are not limited to, benzalkonium chloride, benzethonium chloride, methylparaben, ethylparaben, propylparaben, butylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate or sodium benzoate, potassium sorbate or sodium sorbate, sodium propionate, sorbic acid, etc., and combinations thereof. Exemplary preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, β-carotene, citric acid, ascorbic acid, etc., and combinations thereof. Exemplary buffers for controlling pH may include, but are not limited to, sodium phosphate, sodium citrate, sodium succinate, histidine (or histidine-HCl), sodium malate, sodium carbonate, etc., and / or combinations thereof. Exemplary cryoprotectants include, but are not limited to, mannitol, sucrose, trehalose, lactose, glycerol, dextrose, etc., and combinations thereof. Exemplary diluents such as: lactose, starch, cellulose derivatives, inorganic calcium salts, sorbitol, etc. Exemplary binders such as: starch, gelatin, sodium carboxymethylcellulose, polyvinylpyrrolidone, etc. Exemplary antioxidants such as: vitamin E, sodium bisulfite, sodium sulfite, butylated hydroxyanisole, etc. Exemplary lubricants such as: magnesium stearate, colloidal silicon dioxide, talc, etc. Exemplary disintegrants such as: starch, methylcellulose, xanthan gum, croscarmellose sodium, etc.

[0032] The dosage form of the drug of the present invention can be in the form of an oral preparation, such as tablets, capsules, pills, powders, granules, suspensions, syrups, etc.; or it can be in the dosage form of injection, such as injection solutions, powder for injection, etc., through intravenous, intraperitoneal, subcutaneous or intramuscular routes. All dosage form forms used are well known to those of ordinary skill in the pharmaceutical art.

[0033] On the other hand, the present invention provides the use of the single-domain antibody against NUPR1 or the fusion protein in the preparation of a reagent or kit for detecting NUPR1 in a sample.

[0034] On the other hand, the present invention provides the use of the single-domain antibody against NUPR1 or the fusion protein in the preparation of a drug for preventing and / or treating cancer.

[0035] The cancer includes but is not limited to: pancreatic cancer, breast cancer, liver cancer, lung cancer, colorectal cancer, oral cancer, bladder cancer, myeloma, basal cell carcinoma, cholangiocarcinoma, bone cancer, peritoneal cancer, cervical cancer, cholangiocarcinoma, choriocarcinoma, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, glioblastoma, liver cancer, kidney cancer, laryngeal cancer, leukemia, lymphoma, melanoma, neuroblastoma, ovarian cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, testicular cancer, thyroid cancer, uterine cancer, urinary system cancer, B-cell lymphoma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, hairy cell leukemia, chronic myelogenous leukemia.

[0036] On the other hand, the present invention provides a method for screening a single-domain antibody against NUPR1, the method comprising:

[0037] 1) Replacing the CDR3 domain on the sdAb backbone with a first nucleotide sequence encoding 21 random amino acids, and ligating a nucleotide sequence encoding a first expression tag to the 3'-end of the sdAb backbone to obtain a nucleotide sequence expressing the sdAb library, and then inserting it into a first expression vector to obtain a first recombinant expression vector expressing the sdAb library;

[0038] Preferably, the sdAb backbone is cAbBCII10, and its amino acid sequence is as shown in SEQ ID NO.9;

[0039] 2) Ligating a nucleotide sequence encoding a second expression tag to the 3'-end of the second nucleotide sequence encoding the NUPR1 protein, and then inserting it into a second expression vector to obtain a second recombinant expression vector expressing the NUPR1 protein;

[0040] The first expression tag is different from the second expression tag, and the first expression vector is the same as or different from the second expression vector; preferably, the first expression tag and the second expression tag are selected from Flag or HA; the first expression vector and the second expression vector are selected from pcDNA3.1;

[0041] 3) Co-transfect the first recombinant expression vector and the second recombinant expression vector into cells. After culturing for a period of time, fix the cells, and perform an in situ proximity ligation assay (isPLA) on the fixed cells, and then sort the isPLA-positive cells.

[0042] According to the present invention, the method for screening single-domain antibodies against NUPR1 further includes: 4) Lyse the isPLA-positive cells obtained in step 3) and use them as templates for PCR. Design primers according to the upstream and downstream sequences of the CDR3 domain insertion site of the first recombinant expression vector for PCR amplification, and replace the CDR3 domain on the sdAb backbone with the amplified DNA fragment to obtain NUPR1-specific sdAb.

[0043] According to the present invention, the method for screening single-domain antibodies against NUPR1 further includes: 5) Use the NanoBiT method to verify the NUPR1-specific sdAb obtained in step 4).

[0044] According to the present invention, the method for screening single-domain antibodies against NUPR1 further includes: 6) Link the nucleotide sequence of the sdAb verified to be positive in step 5) to the C-degron (cOdc1) degradation element for further verification.

[0045] According to the present invention, the method for screening single-domain antibodies against NUPR1 further includes: 7) Express, purify and verify the function of the sdAb verified to be positive in step 6).

[0046] Sequence listing of the present invention:

[0047]

[0048]

[0049]

[0050] Advantages of the present invention:

[0051] The present invention screens a #07.81 single-domain antibody against NUPR1, which can specifically bind to NUPR1; after fusing the single-domain antibody with the C-degron degradation element cOdc1, it can achieve targeted degradation of NUPR1 and effectively inhibit the progression of tumors at the same time; after further fusing the TAT transmembrane domain, the single-domain antibody can easily enter cells and minimize immunogenicity.

[0052] At the single-cell level, the present invention utilizes the isPLA technology in combination with next-generation DNA sequencing technology to develop a simple, easy-to-operate, and visual screening method for single-domain antibodies of NUPR1, with low cost and high efficiency.

[0053] In the present invention, "and / or" shall be regarded as a specific disclosure of each of the two designated features or components with or without the other. Thus, the term "and / or" used in a phrase such as "A and / or B" is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" used in a phrase such as "A, B, and / or C" is intended to cover each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0054] "Comprising" and "including" have the same meaning and are intended to be open and allow, but not require, the inclusion of additional elements or steps. When the terms "comprising" or "including" are used herein, the terms "consisting of" and / or "consisting essentially of" are thus also included and disclosed.

[0055] In the present invention, the terms "single-domain antibody", "sdAb", and "VHH" are used interchangeably to refer to an antibody having a single monomeric domain antigen-binding / recognition domain. Such antibodies include camel antibodies or shark antibodies. In some embodiments, VHH contains three CDRs and four framework regions, designated FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In some embodiments, VHH may be truncated at the N-terminus or C-terminus such that it contains only a portion of FR1 and / or FR4, or lacks one or both of these framework regions, as long as VHH substantially maintains antigen binding and specificity.

[0056] As used herein, "percent amino acid sequence identity (%)" and "homology" with respect to a peptide, polypeptide, or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a specific peptide or polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways within the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or MEGALIGNTM (DNASTAR) software. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 . Screening process of anti-NUPR1 sdAbs, wherein:

[0058] A: Workflow for screening anti-NUPR1 sdAbs: 1) Screen CDR3 sequences by isPLA; 2) Verify the CDR3 sequences screened by isPLA using NanoBiT (in vivo live cell interaction) technology; 3) Further verify the CDR3 sequences positive in NanoBiT screening using C-degron experiments; 4) Finally, express and purify the positive sdAbs and verify the functions of the anti-NUPR1 sdAbs.

[0059] B: Verification of NUPR1 CDR3 sequences positive in isPLA screening by NanoBiT experiments: Co-transfect LgBiT-CDR3 and SmBiT-NUPR1 in HEK293T cells and detect the luciferase reporter activity to verify the interaction of different CDRs with NUPR1.

[0060] C: Analysis of the results of C-degron (cOdc1) experiments by immunoblotting (Western blot): Transfect HEK293T cells with expression plasmids of different doses of Flag-sdAb#07.32-cOdc1 (left), Flag-sdAb#07.81-cOdc1 (middle), and Flag-sdAb#31.89-cOdc1 (right) respectively, and compare with the control group (Flag-sdAb-Con), and detect the endogenous NUPR1 protein level, with ACTB as the internal reference.

[0061] D: Transfect MDA-MB-231 cells with expression plasmids of different doses of Flag-sdAb#07.32-cOdc1 (left), Flag-sdAb#07.81-cOdc1 (middle), and Flag-sdAb#31.89-cOdc1 (right) respectively, and compare with the control group (Con or Flag-sdAb-Con), and detect the endogenous protein level of NUPR1, with ACTB as the internal reference.

[0062] E: Transfect MDA-MB-468 cells with expression plasmids of different doses of Flag-sdAb#07.81-cOdc1 and compare with the control group (Flag-sdAb-Con), and detect the endogenous protein level of NUPR1, with ACTB as the internal reference.

[0063] F: Transfect 4T1 cells with expression plasmids of Flag-sdAb#07.81-cOdc1, Flag-sdAb#07.32-cOdc1, and Flag-sdAb#31.89-cOdc1 respectively, and compare with the control group (Con and Flag-sdAb-Con), and detect the endogenous protein level of NUPR1, with ACTB as the internal reference.

[0064] Figure 2 . In vivo and in vitro functional verification results of anti-NUPR1 sdAbs, where:

[0065] A: Purification results of anti-NUPR1 sdAb proteins: Overexpress sdAb-Con-3×Flag-cOdc1-Tat-6×His, sdAb#07.81-3×Flag-cOdc1-Tat-6×His, and sdAb#31.89-3×Flag-cOdc1-Tat-6×His in HEK293F cells, perform protein expression and purification, and use Coomassie blue staining to detect the purification effect, with BSA as a control. S: Supernatant, P: Purified sdAb protein.

[0066] B: Results of immunofluorescence (IF) co-localization analysis of anti-NUPR1 sdAbs in 4T1 cells: Triple immunofluorescence staining of 4T1 cells was performed using anti-Flag mouse antibody (green), anti-NUPR1 rabbit antibody (red), and DAPI (blue), and imaging was performed using a confocal microscope. White arrows indicate the positions of co-localized yellow dots. 4T1 cells were added with purified sdAb-Con, sdAb#07.81, and sdAb#31.89 proteins, and immunofluorescence detection was performed after 48 hours of culture. Scale bar: 20 μm.

[0067] C: Results of GST pull-down experiments to verify the interaction between anti-NUPR1 sdAbs and NUPR1: GST pull-down experiments were performed using purified sdAb-Con or sdAb#07.81, incubated with purified GST or GST-NUPR1 overnight at 4°C, and the bound proteins were separated by SDS-PAGE electrophoresis and analyzed by Coomassie blue staining.

[0068] D: Surface plasmon resonance (SPR) analysis: The left figure shows the SPR sensorgram between sdAb#07.81 and GST, and the right figure shows the SPR sensorgram between sdAb#07.81 and GST-NUPR1.

[0069] E: Verification of the function of anti-NUPR1 sdAb#07.81 by isPLA experiment: 48 hours after transfection, the interaction between anti-NUPR1 and sdAb was detected by isPLA experiment, and white arrows indicate isPLA signal points. Nuclear staining was performed using DAPI (blue), scale bar: 10 μm.

[0070] F-I: Immunoblot analysis results of NUPR1 in MDA-MB-231 and 4T1 cells: After treatment with anti-NUPR1 sdAb #07.81, sdAb #31.89, and the combination of sdAb #31.89 + #07.81 for 48 hours in MDA-MB-231 (F-G) and 4T1 (H-I) cells respectively, the protein expression levels of NUPR1 and Flag-sdAb were detected, with ACTB as the internal reference, and the control group was Con.

[0071] Figures 3A - 3E : Experimental results of anti-NUPR1 sdAb #07.81 inhibiting the progression of 4T1 cells, where:

[0072] Figure 3A : Allograft experiments were conducted on BALB / c mice. Each mouse was injected with 4T1 cells (1×106 cells). The experiment was divided into three groups: control group (Con, n = 7), single-domain antibody control group (sdAb-Con, n = 7), and sdAb #07.81 treatment group (n = 7). Purified sdAb protein was injected into the tumor once every 3 days.

[0073] Figure 3B : Tumor images taken from mice in each group.

[0074] Figure 3C : Tumor weights were measured and statistical analysis was performed using the t-test. **** indicates p < 0.0001.

[0075] Figure 3D : Representative immunohistochemistry (IHC) staining images showed the expression of Ki67 in subcutaneous tumor tissues. Scale bar: 200 μm.

[0076] Figure 3E : The protein levels of Flag-sdAb and NUPR1 in tumors were detected by immunoblot (Western blot), with ACTB as the internal reference control. Detailed implementation methods

[0077] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content recorded in the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the present invention.

[0078] Materials and methods:

[0079] Reagents: DAPI (D9542, Sigma - Aldrich), BCA Protein Assay Kit (23250, Thermo Scientific), BSA (New England BioLabs), ECL (enhanced chemiluminescence) detection reagent (32106, Thermo Fisher Scientific), EB (ethidium bromide, E1385, Sigma - Aldrich), GSH (G2451, Sigma - Aldrich), imidazole (I5513, Sigma - Aldrich), Tris (T1503, Sigma - Aldrich), IgG (AC011, mouse, Abclonal), polyethyleneimine (PEI) (Polysciences, Inc.), Lipo 3000 transfection reagent (Invitrogen, L3000075), Lipo293F transfection reagent (Beyotime, C0518), paraformaldehyde (158127, Sigma - Aldrich), polylysine (P4707, Sigma - Aldrich), protease inhibitor cocktail tablets (4693132001, Roche, Basel, Switzerland), tissue cell lysis buffer (PrimeCell, C1051), restriction enzymes including EcoRI and BamHI (BioLabs, New England), seamless cloning and assembly kit (CU201, TransGen Biotech), DNA Markers Plus II (BM121, TransGen Biotech) and protein prestained Marker (26616, Thermo Scientific).

[0080] Antibodies: Flag antibody (F7425, mouse monoclonal, Sigma - Aldrich), HA antibody (#7695, rabbit monoclonal, Cell Signaling Technology), anti - Flag M2 affinity beads (A2220, Sigma - Aldrich), NUPR1 antibody (rabbit polyclonal, self - made antibody).

[0081] Kits: Protein:Protein Interaction System (N2014, Promega, USA), Duolink In Situ Red Starter Kit Mouse / Rabbit (DUO92101, Sigma-Aldrich, Germany).

[0082] Cells: HEK293T cells, MDA-MB-231 cells and 4T1 cells were purchased from ATCC (Manassas, VA, USA).

[0083] Example 1 Construction of sdAb library

[0084] The nucleotide sequence of a camel-derived sdAb (cAbBCII10, amino acid sequence shown in SEQ ID NO. 9) was synthesized by gene synthesis and cloned into the pcDNA3.1 vector. The CDR3 region sequence was replaced with EcoRI and BamHI restriction sites and a 6-base pair linker DNA sequence (5'-GAATTCGGCAGCGGATCC-3'). The complete nucleic acid sequence is shown in SEQ ID NO. 11, and a 3×Flag tag was ligated to the 3' end of the sdAb gene to obtain the sdAb control (abbreviated as "sdAb Con") plasmid. At the same time, the sdAb Con plasmid is also the backbone sequence for library construction.

[0085] The first nucleotide sequence encoding 21 random amino acids was replaced into the CDR3 region of the sdAb Con plasmid to obtain the first recombinant expression vector expressing the sdAb library. The first nucleotide sequence contains 63 degenerate bases of deoxynucleotides and also contains approximately 20 nucleotides matching the sdAb con at both ends. The first nucleotide sequence is: 5'-CTATTTATTATTGTGCTGCT(NNN) 21 TGGGGTCAAGGTACTCAAGTTACT-3'. At the same time, a primer complementary to its 3' end was synthesized for filling in the DNA double strand by PCR. The primer sequence is: 5'-AGTAACTTGAGTACCTTGACC-3'.

[0086] Example 2 Screening of anti-NUPR1 sdAbs

[0087] 1) isPLA screening

[0088] The first recombinant expression vector expressing the sdAb library with a Flag tag and the second recombinant expression vector expressing NUPR1 with an HA tag were co-transfected into HEK293T cells. After culturing for 48 h, the cells after trypsin digestion were fixed with 1% PFA and then collected for subsequent isPLA. The isPLA experiment was performed according to the requirements of the Duolink In Situ Red Starter Kit Mouse / Rabbit kit (DUO92101, Sigma-Aldrich). The collected cells were permeabilized with PBS containing 0.5% TritonX-100 at room temperature for 10 min, and then the cells were transferred to a 1.5 mL EP tube and mixed with the blocking solution, and blocked at 37 °C for 1 h. Subsequently, anti-HA rabbit antibody and anti-Flag mouse antibody were added and incubated at 37 °C for 1 h. Then, the MINUS (anti-mouse) and PLUS (anti-rabbit) PLA probes provided in the kit were added and incubated at 37 °C for 1 h. The hybridized probes were then added to the Ligation-Ligase solution for ligation at 37 °C for 30 min, and finally, the Amplification-Polymerase solution was added for amplification at 37 °C for 100 min.

[0089] After isPLA, HEK293T cells were washed twice with PBS buffer containing 1% BSA, 2 mM EDTA, and 0.1% NaN3 (1500 rpm, 4 °C, 5 min), sorted immediately with a BD FACS Aria II flow cytometer, and the data were analyzed with FlowJoV10.0.7 software (Tree Star). The sorted positive cells were collected in nucleic acid-free water and used as a PCR template for cDNA amplification.

[0090] Cells collected in nucleic acid-free water by flow sorting were incubated in a metal bath at 95 °C for 10 min and used as templates for PCR to amplify DNA fragments in the CDR3 region. PCR primers CDR3-forward: 5’-ACACCGCCATCTACTACTGC-3’ and CDR3-reverse: 5’-GCTGCTCACTGTCACTTGTG-3’, which are complementary to the upstream and downstream sequences of the CDR3 region, were synthesized by Invitrogen. Phusion Hot Start II High-Fidelity PCR Master Mix (Thermo Scientific) was used according to the instructions. In a 50 μL reaction system, 500 cells were selected as templates, and the final concentration of each primer was 0.5 μM. The PCR procedure included: pre-denaturation at 98 °C for 2 min; 50 cycles of amplification, including denaturation at 98 °C for 30 sec, annealing at 62 °C for 30 sec, and extension at 72 °C for 1 min; and finally continued extension at 72 °C for 10 min. The PCR products were used for the construction of the next round of screening sub-library. If no further screening was carried out, the PCR products were directly used for next-generation sequencing; if monoclonal antibodies were desired, the PCR products could be subcloned into the sdAb Con vector, and the monoclonal antibodies were sequenced after obtaining them. The sequencing primer was 5’-GCACCAAAATCAACGGGAC-3’.

[0091] 2) The NUPR1 sdAb sequences that were positive in the isPLA screening were verified by NanoBiT experiments

[0092] NanoBiT is a two-subunit system based on luciferase and is used to detect protein interactions in living cells. The NanoBiT system consists of two small units: Large BiT (LgBiT; 18 kDa) and Small BiT (SmBiT; 11-amino acid peptide), which are fused to the proteins to be tested, respectively. When there is an interaction between the two proteins to be tested, LgBiT will be complementary to SmBiT in structure, forming a functional luciferase, which will then react with the substrate to produce a bright luminescence signal. According to the empty plasmid provided by the PPI System Technical Manual TM461 Promega kit (Promega, Cat#N2014), the nucleic acid sequence encoding sdAb was ligated to the LgBiT vector, and the resulting plasmid was hereinafter referred to as the LgBiT-CDR3 plasmid. The NUPR1 sequence was ligated to SmBiT, and the resulting plasmid was hereinafter referred to as the SmBiT-NUPR1 plasmid.

[0093] Plate HEK293T cells in 24-well plates. On the next day, co-transfect the LgBiT-CDR3 plasmid and the SmBiT-NUPR1 plasmid. After 6 - 8 hours, aspirate the medium from the 24-well plates and replace it with phenol red-free medium. Detect after 48 hours. First, aspirate the medium, and then slowly rinse once with PBS. The Nano- Live Cell Substrate and Nano- LCS Dilution Buffer (Promega, Cat#N2011) are mixed at a ratio of 1:19, and then added to the plate. Immediately, perform luciferase reporter activity detection using a microplate reader. The results are as shown in Figure 1 Figure B. The results show that compared with the control group sdAb sequence (denoted as "NAb(Con)" in the figure), the sdAb sequences numbered #07.32, #31.53, #31.89, and #07.81 all showed significant luciferase activity. The results indicate that the sdAbs numbered #07.32, #31.53, #31.89, and #07.81 have specific binding to NUPR1 in live cells, and the sdAb of #07.81 has the strongest binding specificity.

[0094] 3) Verify the positive sdAb sequences screened by isPLA and NanoBiT through C-degron experiments

[0095] Transfect the expression plasmids of sdAb#07.32 - 3×Flag-cOdc1, sdAb#07.81 - 3×Flag-cOdc1, and sdAb#31.89 - 3×Flag-cOdc1 into HEK293T, MDA-MB-231, MDA-MB-468, and 4T1 cells respectively, and compare with the control group (sdAb-Con-3×Flag-cOdc1). Detect the endogenous NUPR1 protein level by immunoblotting (Western blot), using ACTB as an internal reference. The specific procedure is as follows: After 48 hours of transfection, collect the cells and treat them in lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% IGEPAL CA-630, 0.2 mM EDTA, pH 8.0), and then centrifuge at 12,000 rpm for 15 minutes. Perform Western blotting with the corresponding antibodies, and then develop using an ECL detection system (32106, Thermo Scientific), expose to an x-ray film, and scan and record the exposed bands. The results are as shown in Figure 1As shown in C-F. The results showed that both sdAb#07.32-3×Flag-cOdc1 (denoted as "#07.32-cOdc1" in the figure) and sdAb#07.81-3×Flag-cOdc1 (denoted as "#07.81-cOdc1" in the figure) could significantly reduce the level of endogenous NUPR1 protein. The sdAb of #07.81 showed high activity in all cell lines. The above results indicated that the sdAb of #07.81 had a strong specific binding ability with NUPR1, and the connection of cOdc1 could achieve the targeted degradation of NUPR1.

[0096] Example 3 Functional verification of anti-NUPR1 sdAbs

[0097] 1) Purification of anti-NUPR1 sdAb protein

[0098] The eukaryotic expression plasmids with secretion peptides, sdAb-Con-3×Flag-cOdc1-Tat-6×His, sdAb#07.81-3×Flag-cOdc1-Tat-6×His and sdAb#31.89-3×Flag-cOdc1-Tat-6×His, were transiently transfected into HEK293F cells and cultured at 37 °C, 130 rpm, and 8% CO2. After 72 h of transfection, the supernatant was collected and filtered through a 0.45 μm filter. Then, the protein was purified using a nickel ion affinity chromatography column (Sangon Biotech, C600793). Then, it was shown by Coomassie brilliant blue staining, with bovine serum albumin (BSA) as a control, to obtain the purified protein, which was denoted as "sdAb-Con", "sdAb#07.81" and "sdAb#31.89" respectively in this example. The results were as Figure 2 shown in A. 2) Immunofluorescence (IF) co-localization analysis of 4T1 cells

[0099] Purified recombinant proteins of sdAb-Con, sdAb#07.81 and sdAb#31.89 were added to 4T1 cells, and immunofluorescence detection was performed after 48 hours of culture. 4T1 cells were triple immunofluorescently stained with anti-Flag mouse antibody, anti-NUPR1 rabbit antibody and DAPI, and imaged by a confocal microscope. The results were as Figure 2 shown in B. The results showed that both sdAb#07.81 and sdAb#31.89 co-localized with endogenous NUPR1 in 4T1 cells, and the co-localization effect of sdAb#07.81 was significantly better than that of sdAb#31.89.

[0100] 3) GST pull-down experiment to verify the interaction between sdAbs and NUPR1

[0101] The GST pull-down assay was performed using purified sdAb-Con or sdAb#07.81, which were incubated with purified GST or GST-NUPR1 overnight at 4 °C. The bound proteins were separated by SDS-PAGE electrophoresis and analyzed by Coomassie Brilliant Blue staining. The results are as Figure 2 shown in C. The results showed that in vitro, sdAb#07.81 could bind to GST-NUPR1 and directly interact, while it did not bind to GST and could not interact. The results indicated that there was a specific binding interaction between sdAb#07.81 and NUPR1.

[0102] 4) Surface plasmon resonance (SPR) analysis

[0103] Purified GST protein, GST-NUPR1 protein, sdAb Con protein, and sdAb#07.81 protein required for the SPR experiment were prepared in advance and ensured that they were all in the same buffer system (1×PBS). The experiment was carried out on a Biacore 8K instrument (Cytiva), and a CM5 chip was selected for the experiment. The CM5 surface was activated by using a prepared mixture of NHS and EDC (1:1, v / v), and sdAb-Con and dAb#07.81 were coupled to the CM5 chip by standard coupling methods. The protein concentration was 20 μg / mL, and the ligand was diluted with a solution with a pH of 4.0 provided by Cytiva before coupling (the choice of pH depends on the isoelectric point of the protein), and the remaining wells were filled with running buffer. Capturing the analyte: The analyte GST and GST-NUPR1 proteins were diluted to the concentrations determined by preliminary experiments: 4000 nM, 2000 nM, 1000 nM, 500 nM, 250 nM, and bound to the chip at a flow rate of 30 μL / min for 180 s, and then dissociated with running buffer for 200 s. After each cycle ended, the chip was regenerated with a glycine solution with a pH of 3.0, and a total of 9 cycles were set up. After the experiment ended, the dissociation constant was calculated and analyzed using Cytiva software. The results are as Figure 2 shown in D. The results showed that in vitro, sdAb#07.81 could bind to GST-NUPR1 and did not bind to GST. The results indicated that there was a specific binding interaction between sdAb#07.81 and NUPR1.

[0104] 5) Verification of the function of the sdAb of #07.81 by isPLA experiment

[0105] After transfecting the plasmid expressing sdAb#07.81-3×Flag into HEK293T cells, the cells were seeded onto the poly-lysine (P4707, Sigma-Aldrich)-coated coverslips in 12-well plates. After overnight culture, the cells on the coverslips were fixed with 1% paraformaldehyde (15812, Sigma-Aldrich) for 10 min, washed twice with PBS buffer, and then subjected to isPLA experiments. Among them, primary antibodies NUPR1 antibody and Flag antibody were used. Finally, the coverslips were sealed with a mounting medium containing DAPI, and the experimental results were analyzed and photographed using a laser scanning confocal microscope. The results are as Figure 2 shown in E. The results showed that compared with the negative control sdAb sequence (sdAb-Con), the sdAb of #07.81 could specifically interact with endogenous NUPR1, and the red fluorescence signal (indicated by the white arrow) was the positive signal. The above results indicated that the sdAb of #07.81 could specifically bind to endogenous NUPR1.

[0106] 6) Immunoblot analysis of anti-NUPR1 sdAb protein in MDA-MB-231 and 4T1 cells

[0107] After treating MDA-MB-231 and 4T1 cells with the purified anti-NUPR1 sdAb#07.81, sdAb#31.89, and the combination of sdAb#31.89+sdAb#07.81 prepared above for 48 hours respectively, the protein expression levels of NUPR1 and anti-NUPR1 sdAb were detected, and ACTB was used as an internal reference. The control group was Con. The results are as Figure 2 shown in F-I. The results showed that compared with the sdAb-Con group, sdAb#07.81 could specifically target NUPR1, and with the increase of protein concentration, its ability to degrade NUPR1 gradually increased. The above results indicated that the purified sdAb#07.81 still had the ability to target NUPR1 and still had the function of degrading NUPR1.

[0108] Example 4 Experimental study on anti-NUPR1 sdAb#07.81 inhibiting 4T1 cells

[0109] The experiments of this example were carried out using the purified protein prepared in Example 3.

[0110] Allogeneic transplantation experiments were conducted on BALB / c mice. 4T1 cells (1×106 cells) were subcutaneously injected into the back of each mouse on the posterior side of one side. The experiments were divided into three groups: control group (Con, n = 7), single-domain antibody control group (sdAb-Con, n = 7), and sdAb#07.81 treatment group (n = 7). On the 5th, 8th, 11th, and 14th days, purified sdAb protein (15 mg / kg) was injected into the tumor once. On the 17th day, the tumors were removed for detection. The tumor images and weights are as Figures 3B - 3C shown. The results showed that compared with the Con group and the sdAb-Con group, the tumor volume and weight of the mice in the sdAb#07.81 group were significantly reduced. The above results indicate that sdAb#07.81 has an obvious inhibitory effect on tumors. The expression of Ki67 in subcutaneous tumor tissues was shown by immunohistochemistry (IHC) staining images, and the results are as Figure 3D shown. The results showed that the positive signal of the Ki67 expression level in the tumor tissues of the sdAb#07.81 group was significantly reduced. The above results indicate that sdAb#07.81 can effectively inhibit the proliferation of tumor cells. The protein levels of anti-NUPR1 sdAb and NUPR1 in the tumors were detected by immunoblotting (Western blot), and ACTB was used as an internal reference control. The results are as Figure 3E shown. The results showed that compared with the Con group and the sdAb-Con group, the expression of the NUPR1 protein level in the sdAb#07.81 group was significantly decreased. The above results indicate that sdAb#07.81 targets NUPR1 and can degrade NUPR1.

[0111] In the present invention, an anti-NUPR sdAb library containing a randomly arranged CDR3 sequence of 21 amino acids was screened by isPLA high-throughput, and a single-domain antibody specifically recognizing NUPR1 - sdAb numbered #07.81 was obtained. By connecting the C-degron degradation element cOdc1 to the single-domain antibody sequence of #07.81, specific degradation of NUPR1 can be achieved, and the function of inhibiting tumor progression can be exerted.

[0112] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A single-domain antibody against NUPR1, characterized in that: The single-domain antibody comprises 3 complementary determining regions, namely HCDR1, HCDR2 and HCDR3. Among them, the amino acid sequences of HCDR1, HCDR2 and HCDR3 have at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequences shown in SEQ ID NO.1 to SEQ ID NO.3 respectively; Preferably, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are respectively as shown in SEQ ID NO.1 to SEQ ID NO.

3.

2. The single-domain antibody according to claim 1, characterized in that: The single-domain antibody further comprises 4 framework regions alternately connected with 3 complementary determining regions, namely FR1, FR2, FR3 and FR4. Among them, the amino acid sequences of FR1, FR2, FR3 and FR4 have at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequences shown in SEQ ID NO.4 to SEQ ID NO.7 respectively; Preferably, the amino acid sequences of FR1, FR2, FR3 and FR4 are respectively as shown in SEQ ID NO.4 to SEQ ID NO.

7.

3. The single-domain antibody according to claim 1, characterized in that: The amino acid sequence of the single-domain antibody has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO.8; preferably, the amino acid sequence of the single-domain antibody is as shown in SEQ ID NO.

8.

4. A fusion protein, characterized in that: The fusion protein comprises the single-domain antibody against NUPR1 according to any one of claims 1-3; Preferably, the fusion protein comprises A-B-C-D-E-F, where A is a secretion peptide or absent, B is the single-domain antibody according to any one of claims 1-3, C is a first tag sequence or absent, D is a degradation element cOdc1 or absent, E is a cell-penetrating peptide or absent, F is a second tag sequence or absent, and there is or is not a flexible linker between A, B, C, D, E and / or F; the amino acid sequence of the degradation element cOdc1 is: SHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV; Preferably, the cell-penetrating peptide is selected from TAT-derived peptides, antennapedia leader peptides, antennapedia peptides, polyarginine peptides; preferably YGRKKRRQRRR, GRKKRRQRRR, KKWKMRRNQFWVKVQRG, RQIKIWFQNRRMKWKK, or a polyarginine peptide containing 6-9 arginines; more preferably YGRKKRRQRRR; Preferably, the first tag sequence and the second tag sequence are the same or different, and are selected from His tag, Myc tag, HA tag, GST tag, mCherry, GFP, Flag tag; preferably, the first tag sequence and the second tag sequence are different, and are respectively His tag and Flag tag; preferably, the first tag sequence is HHHHHH, and the second tag sequence is DYKDHDGDYKDHDIDYKDDDDK, or the first tag sequence is DYKDHDGDYKDHDIDYKDDDDK, and the second tag sequence is HHHHHH; Preferably, the fusion protein is A-B-C-D-E-F, wherein A is a secretion peptide or absent, B is the single-domain antibody according to any one of claims 1-3, C is the first tag sequence or absent, D is the degradation element cOdc1, E is the cell-penetrating peptide, F is the second tag sequence or absent, and there is or is not a flexible linker between A, B, C, D, E and / or F; Preferably, the amino acid sequence of the fusion protein is MGQVQLVESGGGSVQAGGSLRLSCTASGGSEYSYSTFSLGWFRQAPGQEREAVAAIASMGGLTYYADSVKGRFTISRDNAKNTVTLQMNNLKPEDTAIYYCAALSGPSWDWECLSAIVASGELNWGQGTQVTVSSGSGDYKDHDGDYKDHDIDYKDDDDKGSGSHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINVYGRKKRRQRRRHHHHHH; Preferably, the fusion protein is A-B-C-D-E-F, wherein A is a secretion peptide or absent, B is the single-domain antibody according to any one of claims 1-3, C is the first tag sequence, D is absent, E is the cell-penetrating peptide or absent, F is absent, and there is or is not a flexible linker between A, B, C and / or E; Or, Preferably, the fusion protein is A-B-C-D-E-F, wherein A is a secretion peptide or absent, B is the single-domain antibody according to any one of claims 1-3, C is absent, D is absent, E is the cell-penetrating peptide or absent, F is the second tag sequence, and there is or is not a flexible linker between A, B, E and / or F.

5. A polynucleotide, characterized in that: The polynucleotide comprises a polynucleotide encoding the single-domain antibody according to any one of claims 1-3 or the fusion protein according to claim 4; Preferably, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO.12 or SEQ ID NO.

18.

6. An expression vector, characterized in that, Comprising the polynucleotide according to claim 5.

7. A host cell, characterized in that, Comprising the expression vector according to claim 6.

8. A pharmaceutical composition or a kit, characterized in that, The pharmaceutical composition or the kit comprises the single-domain antibody according to any one of claims 1-3 or the fusion protein according to claim 4.

9. Use of the single-domain antibody according to any one of claims 1-3 or the fusion protein according to claim 4 in the preparation of a reagent or kit for detecting NUPR1 in a sample.

10. Use of the single-domain antibody according to any one of claims 1-3 or the fusion protein according to claim 4 in the preparation of a drug for preventing and / or treating cancer; Preferably, the cancer is breast cancer, pancreatic cancer, lung cancer or colorectal cancer.

11. A method for screening single-domain antibodies against NUPR1, characterized in that, The method comprises: 1) Replacing the CDR3 domain on the sdAb backbone with a first nucleotide sequence encoding 21 random amino acids, and ligating a nucleotide sequence encoding a first expression tag to the 3'-end of the sdAb backbone to obtain a nucleotide sequence expressing the sdAb library, and then inserting it into a first expression vector to obtain a first recombinant expression vector expressing the sdAb library; Preferably, the sdAb backbone is cAbBCII10, and its amino acid sequence is shown in SEQ ID NO.9; 2) Ligating a nucleotide sequence encoding a second expression tag to the 3'-end of the second nucleotide sequence encoding the NUPR1 protein, and then inserting it into a second expression vector to obtain a second recombinant expression vector expressing the NUPR1 protein; The first expression tag is different from the second expression tag, and the first expression vector is the same as or different from the second expression vector; Preferably, the first expression tag and the second expression tag are selected from Flag or HA; the first expression vector and the second expression vector are selected from pcDNA3.1; 3) Co-transfecting the first recombinant expression vector and the second recombinant expression vector into cells, fixing the cells after culturing for a period of time, performing an in situ proximity ligation assay (isPLA) on the fixed cells, and then sorting the isPLA positive cells; Preferably, the method further comprises step 4): lysing the isPLA positive cells obtained in step 3) as a template for PCR, designing primers according to the upstream and downstream sequences of the CDR3 domain insertion site of the first recombinant expression vector for PCR amplification, and replacing the CDR3 domain on the sdAb backbone with the amplified DNA fragment to obtain an NUPR1-specific sdAb; Preferably, the method further comprises step 5): verifying the sequence of the anti-NUPR1-specific sdAb obtained in step 4) by using the NanoBiT method; Preferably, the method further comprises step 6): ligating a C-degron (cOdc1) degradation element to the nucleotide sequence of the sdAb verified positive in step 5) for further verification; Preferably, the method further comprises step 7): expressing and purifying the sdAb verified positive in step 6).

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