Nanometer antibody targeting Pirin and application thereof

Abstract

The invention provides a Pirin-targeted nano antibody and application thereof. The nano antibody comprises a complementary determining region CDR which is a CDR1 shown in SEQ ID NO: 1 and a CDR3 shown in SEQ ID NO: 2, or a CDR1 shown in SEQ ID NO: 10 and a CDR3 shown in SEQ ID NO: 11, and an HV2 shown in an amino acid sequence of SEQ ID NO: 3 and an HV4 shown in an amino acid sequence of SEQ ID NO: 4, or an HV2 shown in SEQ ID NO: 12 and an HV4 shown in SEQ ID NO: 13. The KD value of the nano antibody is in a range of 2.0-15nM, the nano antibody has antigen-antibody binding force, the affinity is close to a nanomole level, and the nano antibody can be regarded as a high-affinity antibody; the compound can be developed into a high-specificity diagnostic reagent, and can be used as a therapeutic drug or an immune sensitizer.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a nanobody targeting Pirin and its applications. Background Technology

[0002] Pirin (PIR) is a highly conserved Fe(II)-dependent nucleoprotein belonging to the cupin superfamily. It plays a central regulatory role in various key cellular processes, particularly in tumorigenesis and immune regulation. One key function is transcriptional and apoptosis regulation: Pirin acts as a transcriptional coactivator, regulating NF1 / CTF1 and Bcl-3, thereby influencing DNA replication, transcriptional activation, and apoptosis signaling pathways. Its core role in tumors is as a "brake" on apoptosis. In tumors such as colorectal cancer, Pirin inhibits FAS-mediated apoptosis through a sophisticated dual mechanism: on the one hand, it prevents the binding of the NFκB2 complex (p52-RELB) to the FAS gene promoter, thus inhibiting FAS expression at the transcriptional level; on the other hand, it inhibits upstream kinase NIK-mediated NFκB2 activation and nuclear translocation. Furthermore, Pirin can directly bind to the FAS protein, retaining it in the cytoplasm and preventing its transport to the cell membrane and the assembly of the apoptosis signaling complex. Secondly, it exhibits enzymatic catalytic activity: Pirin possesses quercetin 2,3-dioxygenase activity, enabling it to catalyze the degradation of quercetin, a natural flavonoid compound with anticancer and anti-inflammatory activities. This function links Pirin to the regulation of intracellular redox homeostasis and signaling pathways such as PI3K / Akt. Thirdly, it plays a crucial role in tumorigenesis: Pirin is highly expressed in various malignant tumors, including colorectal cancer, liver cancer, kidney cancer, and glioma, and its high expression level is often associated with poor prognosis. Its core oncogenic mechanism involves constructing the "PIR-NIK-NFκB2-FAS" survival pathway to inhibit FAS-dependent apoptosis. Since FAS-mediated apoptosis is caused by cytotoxic T lymphocytes (CD8+), Pirin plays a crucial role in tumorigenesis. + Pirin is one of the main weapons of T cells to kill tumor cells. High expression of Pirin is equivalent to putting on a "bulletproof vest" for tumor cells, enabling them to effectively evade the surveillance and elimination of the immune system.

[0003] Given Pirin's crucial role in tumors, developing nanobodies targeting Pirin has great potential to become the next generation of tumor diagnostic and therapeutic tools. Summary of the Invention

[0004] To address the aforementioned issues, this invention proposes a nanobody targeting Pirin and its applications, providing a molecular tool for developing novel Pirin inhibitors.

[0005] To achieve the above objectives, embodiments of the present invention provide a nanobody targeting Pirin in a first aspect, the nanobody comprising a complementarity-determining region (CDR) of: CDR1 shown in SEQ ID NO: 1 and CDR3 shown in SEQ ID NO: 2, or CDR1 shown in SEQ ID NO: 10 and CDR3 shown in SEQ ID NO: 11, and an amino acid sequence of HV2 as shown in SEQ ID NO: 3 and an amino acid sequence of HV4 as shown in SEQ ID NO: 4, or HV2 shown in SEQ ID NO: 12 and HV4 shown in SEQ ID NO: 13.

[0006] According to an embodiment of the present invention, a nanobody targeting Pirin has a KD value in the range of 2.0-15 nM, possesses antigen-antibody binding ability, and has an affinity close to the nanomolar level, and can be regarded as a high-affinity antibody.

[0007] This allows the nanobody to be developed into a highly specific diagnostic reagent, enabling the detection of Pirin expression levels in tumor biopsy samples using immunohistochemistry (IHC) or Western blotting (WB). This not only serves as an auxiliary diagnostic biomarker but, more importantly, as a prognostic indicator. Patients with high Pirin expression may have a worse prognosis and may be insensitive to apoptosis-inducing chemotherapeutic drugs or certain immunotherapies. An ELISA kit developed based on this nanobody, due to its high stability and ease of production, can be used for the quantitative detection of Pirin in serum or tissue lysates, providing a new tool for early tumor screening and monitoring of treatment response.

[0008] This allows the nanobody to be used directly as a therapeutic agent. Because nanobodies can effectively enter protein-protein interaction interfaces (including recessed epitopes) that are difficult for traditional antibodies to reach, they can be designed to specifically block the binding of Pirin to NFκB2 or FAS proteins. This blockade will disrupt Pirin's inhibition of the apoptosis pathway, reactivating the FAS-mediated apoptosis program and thus directly killing cancer cells.

[0009] This allows the nanobody to function as an "immunosensitizer." A key challenge in current tumor immunotherapy is that many "cold tumors" (i.e., tumors with low immune cell infiltration and low immunogenicity) do not respond to immune checkpoint inhibitors (such as PD-1 / PD-L1 antibodies). This Pirin-targeting nanobody, by inhibiting Pirin function, can sensitize tumor cells to CD8. +The cytotoxic effects of T cells have become sensitive again. Therefore, combining them with PD-1 / PD-L1 inhibitors holds promise for transforming "cold tumors" into "hot tumors," thereby overcoming immunotherapy resistance and significantly expanding the applicable population and efficacy of immune checkpoint inhibitors. This strategy aligns with the current cutting-edge trend of using nanobodies to modulate the tumor microenvironment to enhance immunotherapy.

[0010] Optionally, the framework region of the nanobody includes FR1, FR2, FR3 and FR4, the amino acid sequence of FR1 is shown in SEQ ID NO: 5 or SEQ ID NO: 14, the amino acid sequence of FR2 is shown in SEQ ID NO: 6 or SEQ ID NO: 15, the amino acid sequence of FR3 is shown in SEQ ID NO: 7 or SEQ ID NO: 16, and the amino acid sequence of FR4 is shown in SEQ ID NO: 8 or SEQ ID NO: 17.

[0011] Optionally, the nanobody comprises a nanobody with an amino acid sequence as shown in SEQ ID NO: 9 or SEQ ID NO: 18.

[0012] According to an embodiment of the present invention, in a second aspect, a nucleic acid molecule is provided, said nucleic acid molecule encoding the above-described nanobody targeting Pirin.

[0013] According to an embodiment of the present invention, a third aspect provides a construct comprising the above-described nucleic acid molecule.

[0014] According to an embodiment of the present invention, in a fourth aspect, the application of the above-described pirin-targeting nanobody in the preparation of antitumor drugs is proposed.

[0015] According to embodiments of the present invention, in a fifth aspect, the above-described pirin-targeting nanobody is proposed for use in the preparation of tumor diagnostic / prognostic reagents, or for the preparation of diagnostic / prognostic kits.

[0016] According to an embodiment of the present invention, in a sixth aspect, the application of the above-described pirin-targeting nanobody in the preparation of a tumor immunosensitizer is proposed.

[0017] According to an embodiment of the present invention, a pharmaceutical composition comprising the above-described nanobody targeting Pirin is provided in a seventh aspect.

[0018] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0019] Figure 1 This is a chromatogram of the phage particle pADL23c from an embodiment of the present invention;

[0020] Figure 2 This is a schematic diagram of the process for constructing a striped bamboo shark natural nanobody phage display library according to an embodiment of the present invention;

[0021] Figure 3 These are the total RNA electrophoresis bands of each striped bamboo shark in this embodiment of the invention, wherein the upper image shows a sample from spleen tissue and the lower image shows a sample from peripheral blood leukocytes.

[0022] Figure 4 The cDNA from the spleen / peripheral blood leukocytes of each striped bamboo shark in this embodiment of the invention is obtained via V NAR Product bands after amplification with specific primers;

[0023] Figure 5 This invention relates to the determination of the phage display library size in an embodiment of the invention, where, from left to right, 10 represent the total size of the library. -7 10 -8 10 -9 ;

[0024] Figure 6 This is a map of the pET28a plasmid, a prokaryotic expression vector according to an embodiment of the present invention.

[0025] Figure 7 This is a schematic flowchart of the process of screening antibody sequences using a phage library according to an embodiment of the present invention;

[0026] Figure 8 These are electrophoretic bands of pIIIa protein in an embodiment of the present invention, wherein: pIIIa(1-438) molecular weight: 48.6 kDa; expression vector ORF expression product: 52.1 kDa; control: expression product not induced by IPTG; 250: pIIIa(1-438) sample eluted with 250 mM imidazole; 500: pIIIa(1-438) sample eluted with 500 mM imidazole;

[0027] Figure 9 The images show the phage enrichment for anti-pIIIa in an embodiment of the present invention, where (a) is a first round of screening and (b) is a second round of screening.

[0028] Figure 10 This is the Phage ELISA validation of anti-pIIIa two-round screening monoclonal antibodies in an embodiment of the present invention. The horizontal axis represents each monoclonal antibody corresponding to the 96-well plate, the vertical axis represents the absorbance ratio of the experimental group to the control group, and the red dashed line represents the judgment criteria for positive monoclonal antibodies (experimental group: control group ≥ 5).

[0029] Figure 11The following are examples of phage enrichment for screening anti-Pirin in embodiments of the present invention, including (a) a first round of screening and (b) a second round of screening.

[0030] Figure 12 This is the Phage ELISA validation of anti-Pirin two-round screening monoclonal antibodies in this embodiment of the invention. The horizontal axis represents each monoclonal antibody corresponding to the 96-well plate, the vertical axis represents the absorbance ratio of the experimental group to the control group, and the red dashed line represents the judgment criteria for positive monoclonal antibodies (experimental group: control group ≥ 5).

[0031] Figure 13 The following are examples of the enrichment of phages against p53OD in the embodiments of the present invention, including (a) a first round of screening and (b) a second round of screening.

[0032] Figure 14 This is the Phage ELISA validation of anti-p53OD two-round screening monoclonal antibodies in this embodiment of the invention. The horizontal axis represents each monoclonal antibody corresponding to the 96-well plate, the vertical axis represents the absorbance ratio of the experimental group to the control group, and the red dashed line represents the judgment criteria for positive monoclonal antibodies (experimental group: control group ≥ 5).

[0033] Figure 15 The following are examples of phage enrichment for screening against DeepVent-VNAR in embodiments of the present invention, including (a) a first round of screening and (b) a second round of screening.

[0034] Figure 16 This is the Phage ELISA validation of anti-DeepVent-VNAR two-round screening monoclonal antibodies in an embodiment of the present invention. The horizontal axis represents each monoclonal antibody corresponding to the 96-well plate, the vertical axis represents the absorbance ratio of the experimental group to the control group, and the red dashed line represents the judgment criteria for positive monoclonal antibodies (experimental group: control group ≥ 5).

[0035] Figure 17 This is a screening method for anti-RBD-V in an embodiment of the present invention. NAR The phage enrichment status, including (a) first round of screening and (b) second round of screening;

[0036] Figure 18 This is an embodiment of the anti-RBD-V method of the present invention. NAR Phage ELISA validation of monoclonal antibodies in the second round of screening. The horizontal axis represents each monoclonal antibody in the 96-well plate, the vertical axis represents the absorbance ratio of the experimental group to the control group, and the red dashed line represents the criteria for judging positive monoclonal antibodies (experimental group: control group ≥ 5).

[0037] Figure 19This is a comparison of positive sequences screened in an embodiment of the present invention, where the top image shows amino acids 1-64; the bottom image shows amino acids 65-128; light blue: CDR1; green: CDR2; red: CDR3; purple: HV2 / HV4; bright yellow: typical cysteine; V H -A2NXP8: Anti-erythrocyte surface antigen-human IgG heavy chain variable region; VHH-A1: Anti-HSPBAP1-camel nanobody; V NAR -D8: Screening for positive anti-pIIIa V antibodies NAR Sequence; V NAR -D4: Screening for positive anti-Pirin V antibodies NAR Sequence; V NAR -D2: Screening for positive anti-p53ODV antibodies NAR Sequence; V NAR -A1: Screening for positive anti-DeepVent V antibodies NAR Sequence; V NAR -A6: Screening for positive anti-RBDV antibodies NAR sequence.

[0038] Figure 20 This is a map of the eukaryotic expression vector pcDNA3.4-hIgG-Fc plasmid according to an embodiment of the present invention;

[0039] Figure 21 V is an embodiment of the present invention. NAR Protein (periplasmic expression) electrophoresis bands, the antigen name is following the "anti_" symbol; for example, anti_pIIIa indicates anti-pIIIa-V. NAR "Control" refers to the control group that was not induced by IPTG; BSA represents bovine serum albumin, used to determine sample concentration; (a) anti-pIIIa-V NAR (a) Expression of anti-p53OD-V; (b) Anti-p53OD-V NAR and some Pirin-V NAR (c) partial anti-Pirin-V expression; NAR (d) Anti-RBD-V expression; NAR and anti-DeepVent-V NAR The expression.

[0040] Figure 22 V is an embodiment of the present invention. NAR In eukaryotic protein electrophoresis bands, the antigen name is following the "anti_" symbol; for example, anti_pIIIa indicates anti-pIIIa-V. NAR BSA stands for bovine serum albumin, used to determine sample concentration; (a) anti-pIIIa-V NAR Anti-Pirin-V NAR Anti-p53OD-VNAR (a) Expression of anti-RBD-V NAR Anti-DeepVent-V NAR The expression.

[0041] Figures 23-27 This is a binding curve of the antigen-antibody affinity assay for BLI according to an embodiment of the present invention. The horizontal axis represents the time elapsed during the experiment, and the vertical axis represents the signal intensity. In this experiment, 0-500s represents the antigen-antibody affinity assay. NAR During the binding phase, 500-1100s is the antigen-V phase. NAR During the dissociation phase, for each antigen-V NAR Yes, experiments were conducted using three to four concentration gradients. The black curve represents the original data, and the red curve represents the fitted curve. Detailed Implementation

[0042] The technical solution of the present invention is illustrated below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not preclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not for limiting the order of the method steps or defining the scope of the present invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the present invention.

[0043] To better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the invention to those skilled in the art.

[0044] Culture medium and reagent formulations:

[0045] 2×YT culture medium: 1.6% w / v tryptone, 1% w / v yeast extract, 0.5% w / v sodium chloride, with ultrapure water as solvent. Stir well and autoclave at 121℃ for 20 min. When preparing solid culture medium, add 1.5% w / v agar powder before adding ultrapure water.

[0046] TB culture medium: tryptone 1% w / v, yeast extract 1.2% w / v, glycerol 2% v / v, with ultrapure water as solvent, stir well, and autoclave at 121℃ for 20 min. When preparing solid culture medium, add 1.5% w / v agar powder before adding ultrapure water.

[0047] Tris-EDTA(TE): Tris-HCl 10mM, EDTA 1mM, NaOH 1mM, ultrapure water as solvent, stir well, adjust pH to 8.0 with HCl, autoclave at 121℃ for 20min.

[0048] Guanidine hydrochloride solution (5M): 5M guanidine hydrochloride, using ultrapure water as solvent, is stirred until homogeneous.

[0049] New Wash: NaCl 0.2M, EDTA 2mM, Tris-HCl 15.4mM, use ultrapure water as solvent, stir well, adjust pH to 7.5 with HCl, autoclave at 121℃ for 20min.

[0050] The preparation process of helper phage M13KO7 is as follows:

[0051] (1) Resuscitate TG1 strain and streak it on a solid culture medium plate.

[0052] (2) Pick single clones and inoculate them into 5 mL of YT culture medium. Incubate on a shaker at a constant temperature until OD. 600 =0.5 (37℃, 200rpm), add helper phage M13KO7 (10 14 CFU was cultured in a constant temperature shaker (37℃, 1h, 200rpm) and then streaked onto a solid culture medium plate (50μg / mL kanamycin sulfate).

[0053] (3) Pick single clones and inoculate them into 5 mL of YT culture medium (50 μg / mL kanamycin sulfate), and incubate in a constant temperature shaker until OD. 600 =1.0 (37℃, 200rpm), transfer to 2L YT culture medium (50μg / mL kanamycin sulfate), and incubate overnight on a constant temperature shaker (25℃, 200rpm).

[0054] (4) The subsequent phage purification steps are the same as described above, yielding a final concentration of 5.0 × 10⁻⁶. 13 A helper phage solution with cfu / mL.

[0055] 5×TEDA: Tris-HCl 0.5M, DTT 50mM, MgCl2 50mM, PEG8000 2.5% w / v, NEBT5Exonuclease M0363S 10U / mL 0.1% w / v, with ultrapure water as solvent, stirred until homogeneous.

[0056] Buffer A (His-tagged protein purification): Tris-base 25mM, imidazole 25mM, NaCl 150mM, glycerol 10% v / v, ultrapure water as solvent, stir well, adjust pH to 7.5 with HCl, autoclave at 121℃ for 20min.

[0057] Buffer B (His-tagged protein purification): Tris-base 25mM, imidazole 500mM, NaCl 150mM, glycerol 10% v / v, ultrapure water as solvent, stir well, adjust pH to 7.5 with HCl, autoclave at 121℃ for 20min.

[0058] TES: Tris-HCl 200mM, EDTA 0.5mM, sucrose 500mM, ultrapure water as solvent, stir well, adjust pH to 8.0 with HCl, autoclave at 121℃ for 20min.

[0059] TES / 4: TES 25% v / v, ultrapure water 75% v / v, mixed thoroughly.

[0060] 5×SDS electrophoresis buffer: Tris-base 125mM, glycine 0.96M, SDS 17.3mM, ultrapure water as solvent, stirred evenly, pH adjusted to 8.0 with HCl, autoclaved at 121℃ for 20min.

[0061] 5×SDS Loading Buffer: Tris-HCl 0.25M, SDS 10% w / v, bromophenol blue 0.5% w / v, 41.67% v / v, 5M DTT 10% v / v, with ultrapure water as solvent, stirred evenly, pH adjusted to 8.0 with HCl, and autoclaved at 121℃ for 20 min.

[0062] Binding Buffer (Fc-tag protein purification): NaCl 0.15M, Na2HPO4 20mM, use ultrapure water as solvent, stir well, adjust pH to 7.0 with HCl, filter sterilize with a 0.22μM filter membrane, and store at 4℃.

[0063] Elution Buffer (Fc-tag protein purification): 0.1M glycine, ultrapure water as solvent, stirred until homogeneous, pH adjusted to 3.0 with HCl, sterilized by filtration through a 0.22μM filter membrane, and stored at 4℃.

[0064] Neutralization Buffer (Fc-tag protein purification): Tris-HCl 1M, with ultrapure water as solvent, stir well, adjust pH to 3.0 with HCl, filter sterilize with a 0.22μM filter membrane, and store at 4℃.

[0065] Acid washing reagent: 10mM glycine, using ultrapure water as solvent, stir well, adjust pH to 1.35 with HCl, filter insoluble matter through a 0.45μM filter membrane.

[0066] The formulations of SDS-PAGE protein gels are shown in Table 1 and Table 2 below.

[0067] Table 1. SDS-PAGE separating gel formulation (5 mL)

[0068]

[0069] Table 2. SDS-PAGE Stacking Gel Formulation (2 mL)

[0070]

[0071] The primers used to construct the library in Example 1 are shown in Table 3 below.

[0072] Table 3 is used to obtain V. NAR Amplifier and primer sequence for pADL23c.

[0073]

[0074] The plasmid map of phage particle pADL23c in Example 1 is as follows: Figure 1 As shown, construct V NAR When using the document library, V NAR The amplicon sequence is inserted between two BglI restriction sites, and is in the same reading frame as the PelB leader peptide and the G3 gene.

[0075] The plasmid map of the prokaryotic expression vector pET28a in Example 2 is shown below. Figure 6 As shown.

[0076] The primer sequences used in Example 2 are shown in Table 4 below.

[0077] Table 4 Primer sequences used in Example 2

[0078]

[0079]

[0080] The image of the eukaryotic expression vector pcDNA3.4-hIgG-Fc plasmid in Example 3 is shown below. Figure 20 As shown.

[0081] The primer sequences used in Example 3 are shown in Table 5 below.

[0082] Table 5 Primer sequences used in Example 3

[0083]

[0084] The primers used in Example 4 are shown in Table 6 below.

[0085] Table 6 Primers used in Example 4

[0086]

[0087] The present invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.

[0088] Example 1: Construction of a natural nanobody phage display library from striped bamboo shark

[0089] The library construction process in this embodiment is as follows: Figure 2 As shown. Striped bamboo shark samples were purchased from the Eighth Seafood Market in Xiamen, Fujian Province. Sharks weighing between 300g and 400g were selected, and 1 to 2 sharks were processed daily, for a total of 20 sharks. Spleen tissue rich in lymphocytes and peripheral blood leukocytes were selected for processing. 7.5mL to 15mL of peripheral blood was extracted from each shark.

[0090] 1. Extraction of total RNA

[0091] For each mL of tissue homogenate, the following steps were performed to extract total RNA.

[0092] (1) Add 200 μL of chloroform to the centrifuge tube, shake well, let stand for 10 min, and centrifuge (12000×g, 10 min, 4℃).

[0093] (2) Transfer the upper liquid phase to a new centrifuge tube, add 500 μL of isopropanol, shake gently, let stand at room temperature for 10 min, and centrifuge (12000×g, 10 min, 4℃).

[0094] (3) Use a pipette to remove the supernatant, add 1 mL of 4℃ ethanol, and centrifuge (12000×g, 10 min, 4℃).

[0095] (4) Use a pipette to remove the supernatant and let it stand at room temperature for 10 minutes to allow the residual ethanol to evaporate completely.

[0096] (5) Add 40 μL of RNase-free water to each tube to dissolve the RNA precipitate. Measure the RNA concentration using NanoDrop. On average, 4 mg of total RNA could be extracted from each striped bamboo shark spleen, and 400 μg of total RNA could be extracted from each striped bamboo shark peripheral blood. Verify RNA extraction using agarose gel electrophoresis. Figure 3As shown, three main bands are visible: 28S rRNA, 18S rRNA, and 5S / 5.8S rRNA bands, indicating that total RNA was extracted.

[0097] 2. Reverse transcription of RNA

[0098] Total RNA was reverse transcribed using the ABScript II cDNA First-Strand Synthesis Kit. The mixed reverse transcription reaction system is shown in Table 7 below:

[0099] Table 7 Reverse transcription reaction system (single reaction)

[0100]

[0101] After the above reaction system was mixed evenly, it was placed in a PCR instrument for reverse transcription. The reaction program was set as follows: 42℃, 60 min; 80℃, 5 min.

[0102] 3. cDNA purification using magnetic beads

[0103] (1) Using DNA sorting magnetic beads ( The cDNA was washed using DNA Selection Beads. Before use, the Hieff NGS DNA selection beads were taken out of the refrigerator at 4°C, mixed with a pipette, equilibrated at room temperature for 30 minutes, and then an 80% ethanol solution was prepared.

[0104] (2) Mix the magnetic beads to form a homogeneous turbid solution. Add an equal volume of the magnetic bead turbid solution according to the volume of the reverse transcribed cDNA product. Mix well by pipetting. Aliquot 700 μL into each 1.5 mL centrifuge tube and let stand at room temperature for 5 min. Perform the following operations on each centrifuge tube.

[0105] (3) Briefly centrifuge the centrifuge tube, place it on the centrifuge tube magnetic rack and let it stand for 5 minutes, then discard the supernatant.

[0106] (4) Keep the centrifuge tube still, add 500 μL of 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 seconds, carefully remove the supernatant, repeat this step twice, open the centrifuge tube cap and dry at room temperature until the magnetic bead clumps crack (about 5 minutes).

[0107] (5) Add 170 μL of RNase-free H2O, mix by blowing and sucking, and dissolve at room temperature for 5 min.

[0108] (6) Place the centrifuge tube on the centrifuge tube magnetic rack and let it stand for 5 minutes. Then, transfer the supernatant into a new 1.5 mL centrifuge tube.

[0109] 4. Obtain V by PCR NARSequence Library

[0110] (1) Using the cDNA purified from magnetic beads as a template, PCR was performed using Adley 2×F8 FastLong PCR MasterMix enzyme to achieve V NAR The target fragment was amplified, and the reaction system is shown in Table 8. V NAR The reaction procedure is shown in Table 9:

[0111] Table 8 PCR Reaction System (Single Reaction)

[0112]

[0113] Table 9 VNAR Amplification Reaction Procedure

[0114]

[0115] (2) PCR products were separated by agarose gel electrophoresis, and the target band was recovered using the SteadyPure DNA Gel Recovery Kit. PCR products were then detected using agarose gel electrophoresis, such as... Figure 4 As shown, the band located at 350bp is visible, and it is related to V. NAR The lengths are consistent, indicating that the PCR product is a VNAR fragment. Furthermore, bands smaller than 100 bp are visible, suggesting non-specific amplification or primer dimers.

[0116] 5. Construction and transformation of phage display particles

[0117] V obtained by PCR was purified using an electrophoresis gel extraction kit. NAR The fragments were extracted using DH5α strain to obtain pADL23c phage particles. Both were digested with BglI restriction enzyme, and the fragments were recovered by electrophoresis gel extraction, yielding 56 μg of pADL23c linearized phage particles with BglI sticky ends and 20 μg of V phage particles with BglI sticky ends. NAR Fragments. The pADL23c linearized phage and V were ligated using the T4 ligation kit. NAR The DNA fragments were used entirely in the ligation system, and after ligation, 40 μg of ligated phage particle library was obtained by gel recovery.

[0118] After preparing 5 mL of TG1 strain electroporation competent cells, the competent cells and phage plasmid library were mixed and transformed using 100 electroporation cuvettes with a 1 mm pore size. The bacterial culture was collected, and after a brief recovery period, a small amount of the bacterial culture was diluted to obtain 10% of the total bacterial culture. -6 10 -7 10 -8 10 -9The bacterial suspension was evenly spread onto four circular 2×YT solid plates (ampicillin-resistant) as a library counting plate. The remaining bacterial suspension was evenly spread onto 30 square 2×YT solid plates (all 50cm squares, ampicillin-resistant). Ten of these plates... -6 The plate colonies are too dense, 10 -7 The plate had approximately 850 colonies, 10 -8 The plate had 80 colonies and 10 colonies. -9 The plate has 9 colonies, such as Figure 5 As shown.

[0119] 6. Packaging and purifying the phage display library

[0120] (1) After electroporated bacterial cells were cultured overnight on plates, the plate colonies were collected and preserved. 10 mL of 2×YT culture medium was added to each square plate, and colonies were scraped off using a plastic cell scraper and collected in Erlenmeyer flasks, with a final total volume of 500 mL. The plates were then incubated on a shaker at 37℃ for 45 min at 200 rpm. The bacterial culture was diluted 100-fold, and the OD was measured. 600 The concentration was 0.75. 50 mL of bacterial solution was used to extract bacteriophages, and the remaining bacterial solution was stored at -80°C with glycerol at a final concentration of 25%.

[0121] (2) Resuscitate the above 50 mL bacterial suspension, add 50 mL of bacterial suspension to 6 L of 2×YT (100 μg / mL ampicillin) culture medium, and incubate on a shaker at a constant temperature until OD. 600 =0.5 (37℃, 200rpm).

[0122] (3) To package the phage display library, helper phage M13KO7 (3.0 × 10⁻⁶) was added to the bacterial culture. 14 CFU was used to incubate the bacteria on a shaker at 37°C for 1 hour at 200 rpm to allow helper phages to infect the strains. Kanamycin sulfate was added to a final concentration of 50 μg / mL to screen successfully infected cells, and the bacteria were incubated overnight on a shaker at 25°C at 200 rpm.

[0123] (4) To extract phages, centrifuge to settle the bacterial cells (9000×g, 20 min, 4℃), collect the supernatant, add 1.2 L of PEG6000, incubate on a shaker (4℃, 1 h, 90 rpm), and centrifuge again to settle the phages (10000×g, 40 min, 4℃). Discard the supernatant, resuspend the precipitate in 10 mL of 1×PBS to obtain the phage display library. Dilute the resuspended solution 10 times and measure the OD. 268 Based on the measured values, according to the formula

[0124] bacteriophage number = OD 268 ×5×10 12 ;

[0125] Add PBS to bring the phage count to 1.0 × 10⁻⁶ per mL of solution. 14 The phage display library was aliquoted into 1.5 mL tubes (1 mL per tube) and stored at -80°C. The calculated result was 4.0 × 10⁻⁶. 15 CFU phage.

[0126] Example 2 Specific V NAR filter

[0127] The document filtering process in this embodiment is as follows: Figure 7 As shown.

[0128] (a) Preparation of antigens

[0129] 1. Expression of antigen pIIIa:

[0130] (1) Construction of BL21 intracellular expression vector

[0131] IIIa protein was expressed using intracellular expression of BL21.

[0132] The coding region of the IIIa gene was amplified using the primer pair IIIa lite to 28a F / IIIa lite to 28a F and then ligated between the restriction endonuclease sites BamHI and XhoI of the pET28a expression vector.

[0133] (2) Intracellular expression and purification of Escherichia coli BL21 strain

[0134] Taking the expression of His-tag protein using 200 mL of bacterial culture as an example.

[0135] ① Inoculate a single clone of the BL21 strain transformed with the expression vector into 200 mL of 2×YT culture medium and incubate in a constant temperature shaker until OD. 600 =0.5 (25℃, 200rpm), add IPTG to a final concentration of 0.5mM, and incubate in a constant temperature shaker (25℃, 16h, 200rpm).

[0136] ② Centrifuge to settle the bacterial cells (5000×g, 5min, 4℃), discard the supernatant. Resuspend the bacterial cells in 50mL Buffer A, transfer to a metal ultrasonic disruption cuvette, and place on ice. Use a 250W ultrasonic disruptor at 36% power, sonicating for 3s every 5s, for a total of 40min.

[0137] ③ Centrifuge to settle bacterial cell fragments (12000×g, 30min, 4℃), retain a small amount of sample, and transfer the remaining supernatant to a new centrifuge tube. Take Ni-NTAresin, mix well, take 400μL, centrifuge to settle (5000×g, 30s), discard the supernatant (protective solution), resuspend in 1mL PBS, centrifuge to settle (5000×g, 30s), discard the supernatant (wash with PBS), resuspend in 1mL PBS, add to the supernatant of the sonicated bacterial culture, and rotate using a rotary mixer (2h, 4℃) to allow the protein to bind with Ni-NTA resin.

[0138] ④ Take a protein purification column, pass the supernatant of the mixed bacterial culture through the column, and then rinse the purification column with 5 column volumes of Buffer B. Prepare a mixture of 1.5 mL Buffer A and 1.5 mL Buffer B (containing 250 mM imidazole), plug the bottom of the purification column, transfer it into the purification column, and use a rotary mixer to elute the protein (30 min, 4℃). Collect the eluent (250 mM imidazole). Plug the bottom of the purification column again, transfer 1 mL of Buffer B into the purification column, and use a rotary mixer to elute the protein a second time (30 min, 4℃). Collect the eluent (500 mM imidazole).

[0139] The results are as follows Figure 8 As shown, amino acid fragments 1-438 of the pIIIa protein were extracted and constructed into the pET28a vector for intracellular expression in *E. coli*. The expression product was verified by SDS-PAGE gel electrophoresis, and the results showed successful expression of the pIIIa protein fragment. The amino acid sequence of the pIIIa protein is shown in SEQ ID NO: 19.

[0140] 2. The Pirin antigen is the Pirin protein (Ma H, Cao T, Zhang F, Sun D, ​​Chen L, Lin Y, Lai S, Jiang B, Zhou Y, You J, Liu X, Wang Y, Lin F, Liu Y, Wang J, He W, Li Q. Nuclear Pirin promotes HCC by acting as a key inflammation-facilitating factor, Gut. 2025 Jun 27:gutjnl-2024-334087.) kindly donated by Professor Li Qinxi's laboratory at Xiamen University.

[0141] 3. The p53OD antigen is the p53OD protein with the amino acid sequence shown in SEQ ID NO: 20.

[0142] 4. The DeepVent antigen is the DeepVent protein with the amino acid sequence shown in SEQ ID NO: 21.

[0143] 5. The RBD antigen is the RBD protein with the amino acid sequence shown in SEQ ID NO: 22.

[0144] (II) Screening

[0145] 1. Preparations before screening:

[0146] (1) Activation of TG1 cells. Take the frozen E. coli TG1 strain, streak it on a plate, pick a single colony and inoculate it into 5 mL of 2×YT culture medium, and incubate it overnight at 37℃ in a shaker (200 rpm) as the strain.

[0147] (1) Coating the immunoassay tubes. Take two 5 mL immunoassay tubes and label them as experimental tubes and control tubes. Dissolve 100 μg of antigen protein in 3 mL of 1×PBS and add it to the experimental tube. Prepare 5% skim milk-PBS and add 5 mL to the control tube. Incubate overnight at 4°C.

[0148] 2. Phage bank immune screening:

[0149] (1) Take 1 mL of the striped bamboo shark natural nanobody phage display library (obtained in Example 1), add 2 mL of 5% skim milk-PBS, and mix using a rotary mixer. Empty the experimental tube and control tube, wash three times with 1×PBS, fill with 5% skim milk-PBS, and let stand at room temperature for 1 h.

[0150] (2) Empty the control tube, wash it once with 1×PBS, add the milk-phage library mixture to the control tube, and let it stand at room temperature for 1 hour.

[0151] (3) Empty the experimental tube, wash it three times with 1×PBS, transfer the milk-phage library mixture in the control tube to the experimental tube, wash the empty control tube three times with 1×PBS, fill it with 1×PBS, and let it stand at room temperature for 1 hour.

[0152] (4) Seal the experimental tubes with sealing film and rotate them using a rotary mixer. Inoculate 200 μL of the bacterial culture into 20 mL of 2×YT and incubate on a shaker at a constant temperature until OD. 600 =0.5 (37℃, 200rpm), transfer to 4℃ for later use.

[0153] (5) Empty the experimental tube and control tube, and wash with 1×PBST for 6 times, 5 min each time, and wash with 1×PBS for 2 times, 5 min each time.

[0154] (6) Add 1 mL of 0.1 M HCl to the experimental tube and the control tube, rotate at room temperature for 8 min to wash out the phage, and add 0.5 mL of 1 M Tris-HCl (pH 7.4) to neutralize the pH value.

[0155] (7) Take 20 mL of bacterial solution from 4℃, divide it into two 10 mL bottles, add the contents of the experimental tube and the control tube respectively, and incubate in a constant temperature shaker (37℃, 1h, 200rpm).

[0156] (8) Take a small amount of bacterial samples from both the experimental group and the control group, and dilute them to 10% of the total amount. -4 and 10 -5 Spread evenly on solid culture plates (100 μg / mL ampicillin), incubate overnight at 37°C, and use for phage counting.

[0157] (9) The remaining bacterial cultures of the experimental group and the control group were centrifuged and concentrated, and then evenly spread onto two square solid culture plates with a side length of 50cm (100μg / mL ampicillin) and incubated overnight at 37℃.

[0158] 3. Preparation of bacteriophages after screening:

[0159] (1) Add 10 mL of 2×YT culture medium to each square plate, scrape off the colonies using a plastic cell scraper, dilute 20 times, and measure the OD of the bacterial culture. 600 Inoculate the bacterial culture into 100 mL of 2×YT culture medium (100 μg / mL ampicillin) according to the measured values ​​to achieve the initial OD. 600 =0.1, incubate in a constant temperature shaker until OD 600 =0.5 (37℃, 200rpm).

[0160] (2) Add 5.0×10 12 CFU helper phage M13KO7 was cultured on a shaker for 1 hour (37°C, 200 rpm). Kanamycin sulfate was added to a final concentration of 50 μg / mL, and the mixture was cultured on a shaker overnight (25°C, 200 rpm).

[0161] (3) To extract phages, centrifuge to settle the bacterial cells (9000×g, 20 min, 4℃), collect the supernatant, add 330 mL of PEG6000, incubate on a shaker (4℃, 1 h, 90 rpm), and centrifuge again to settle the phages (10000×g, 40 min, 4℃). Discard the supernatant, resuspend the precipitate in 1 mL of 1×PBS to obtain the screened and enriched phage display library. Measure the OD of the resuspended solution. 268 Calculate the phage concentration and store at -80℃.

[0162] 4. Second / Third Round Screening:

[0163] In the second round of screening, experimental tubes coated with 50 μg of antigen protein were used, and 10 phage libraries enriched after the first round of screening were collected. 12 CFU was used for the second round of screening. For the third round of screening, 25 μg of antigen protein was coated onto experimental tubes, and 10 μg of the phage library enriched after the second round of screening was used. 11 CFU is used for three rounds of screening. The remaining operations are the same as described above.

[0164] 5. Phage ELISA

[0165] (1) Cultivation and screening of positive phages. Add 800 μL of 2×YT culture medium (100 μg / mL ampicillin) to each well of a 96-well deep-well plate. Take the colony counting plate from the last round of screening, pick single colonies from it and inoculate them into the deep wells, one colony per well. Incubate on a shaker until OD. 600 =0.5 (37℃, 200rpm). Take 5.0 × 10 12 CFU helper phage M13KO7 was added to a final volume of 10 mL with 2×YT medium. After mixing, 100 μL of the mixture was added to each well of a deep-well plate and incubated on a shaker at 37°C for 1 h at 200 rpm. Alternatively, 100 μL of kanamycin sulfate solution (50 μg / μL) was added to a final volume of 10 mL with 2×YT medium. After mixing, 100 μL of the mixture was added to each well of a deep-well plate and incubated overnight on a shaker at 25°C at 200 rpm.

[0166] (2) Coating ELISA plates. Prepare two 96-well ELISA plates, one for experimental use and one for control. Take 50 μg of antigen protein, add PBS to a final volume of 10 mL, mix well, and add 100 μL of the mixture to each well of the experimental plate. Add 300 μL of 5% skim milk-PBS to each well of the control plate. Incubate both the experimental and control plates at 4°C overnight.

[0167] (3) Centrifuge the bacterial cells in the 96-well culture plate (1000×g, 20min, 4℃) and store at 4℃. Add PBST to the plate washer to clean the ELISA plate.

[0168] (4) Clean the experimental plate and control plate once with a plate washer, add 300 μL of 5% skim milk-PBS to each well, and incubate at 37°C for 2 hours.

[0169] (5) Clean the experimental plate and control plate once with a plate washer. Add the supernatant of the deep space plate to the experimental plate and control plate one by one according to the well position, adding 100 μL of supernatant to each well. Incubate on a shaker at room temperature for 2 h (90 rpm).

[0170] (6) Wash the experimental plate and control plate 9 times with a plate washer, prepare 1% skim milk-PBS, add anti-M13Bacteriophage antibody HRP (20000×), mix well, add 100μL to each well of the experimental plate and control plate, and incubate at room temperature for 1h (90rpm) on a shaker.

[0171] (7) Clean the experimental plate and control plate 9 times with a plate washer. Add 100 μL TMB colorimetric solution to each well of the experimental plate and control plate. When there is an obvious color change, immediately add 100 μL 0.1M sulfuric acid to terminate the colorimetric reaction.

[0172] (8) Use an enzyme-linked immunosorbent assay (ELISA) reader to read the OD values ​​of the experimental and control plates. 450 As a result, the well numbers of the experimental group and the control group with a reading ratio greater than 5 were recorded, and the bacterial culture in the deep well plate with the corresponding number was sent for sequencing (primer: phiS2-F). The result is the sequence obtained by screening.

[0173] When pIIIa was used as the antigen for screening, there was a significant difference in the number of monoclonal antibodies between the experimental group and the control group after the first round of screening; after the second round of screening, the positive clones in the experimental group were further enriched. Figure 9 ). Because the control group was only 10 -4 The appearance of a single clone on the counting chamber indicates that non-specific antibodies have been screened out. To avoid loss of antibody diversity, a third round of screening will not be performed; instead, the single clones generated in the second round of screening will be used for Phage ELISA validation. From the second round of screening experimental group 10... -5 Ninety-six single clones were selected from the counting chamber and cultured for phage ELISA validation. Of these, 64 single clones met the positive criteria for the phage ELISA experiment: absorbance >5 in the experimental / control group. Figure 10 The monoclonal antibodies were arranged in descending order of experimental / control group, and the top 20 monoclonal antibodies were selected for sequencing and subsequent validation.

[0174] V targeting pIIIa NAR The amino acid sequences involved are shown in Table 10.

[0175] Table 10

[0176]

[0177] When using Pirin as the antigen for screening, as shown in the counting plates, after two rounds of screening, the ratio of monoclonal counts in the experimental group to the control group was significantly higher than that in the first round, with the control group having only 10. -4 The counting chamber has one single clone. Figure 11From the experimental group counting chambers selected in the second round of screening, 96 monoclonal antibodies were randomly selected for Phage ELISA validation, and 23 monoclonal antibodies that met the positive criteria were obtained. Figure 12 All samples were sequenced and subsequently validated.

[0178] V targeting Pirin NAR The amino acid sequences involved are shown in Table 11.

[0179] Table 11

[0180]

[0181] When p53OD was used as the antigen for screening, according to the counting plates, after one round of screening, the number of monoclonal antibodies on the control group plate was higher than that on the experimental group plate. After two rounds of screening, the number of monoclonal antibodies on the experimental group plate was higher than that on the control group plate, indicating that specific antibody enrichment ( Figure 13 From the experimental group counting chambers selected in the second round of screening, 96 monoclonal antibodies were randomly selected for Phage ELISA validation, and 77 monoclonal antibodies that met the positive criteria were obtained. Figure 14 The monoclonal antibodies were arranged in descending order of experimental / control group, and the top 20 monoclonal antibodies were selected for sequencing and subsequent validation.

[0182] V targeting p53OD NAR The amino acid sequences involved are shown in Table 12.

[0183] Table 12

[0184]

[0185] When DeepVent was used as the antigen for screening, the control group showed no monoclonal antibodies in both rounds of screening, while the experimental group showed slight enrichment. Figure 15 From the experimental group counting chambers selected in the second round of screening, 96 monoclonal antibodies were randomly selected for Phage ELISA validation, and 90 monoclonal antibodies that met the positive criteria were obtained. Figure 16 The monoclonal antibodies were arranged in descending order of experimental / control group, and the top 20 monoclonal antibodies were selected for sequencing and subsequent validation.

[0186] V targeting DeepVent NAR The amino acid sequences involved are shown in Table 13.

[0187] Table 13

[0188]

[0189] When RBD was used as the antigen for screening, according to the counting plates, after one round of screening, the control group plate had no monoclonal antibodies; following the experience that screening generally requires two or more rounds, after a second round of screening, the control group plate still had no monoclonal antibodies, while the experimental group plate had a smaller number of monoclonal antibodies. Figure 17 From the experimental group's counting chamber and large square plates selected in the second round of screening, 96 monoclonal antibodies were randomly selected for Phage ELISA validation, and 86 monoclonal antibodies that met the positive criteria were obtained. Figure 18 The monoclonal antibodies were arranged in descending order of experimental / control group, and the top 20 monoclonal antibodies were selected for sequencing and subsequent validation.

[0190] V targeting RBD NAR The amino acid sequences involved are shown in Table 14.

[0191] Table 14

[0192]

[0193] Some of the above-mentioned positive V samples NAR Sequence and V H H, V H Perform sequence alignment ( Figure 19 The following conclusions can be drawn: V H With V H Similar to the H sequence, and V NAR There are significant differences; V NAR Missing CDR2 region; V NAR Although the HV2 / HV4 region is involved in antigen recognition, its sequence is highly conserved. Among the three types of antibody fragments, the CDR3 region has the highest diversity and varies in length, while the CDR1 region has high diversity but consistent length. All three types of antibody fragments have typical cysteine ​​residues and can form conserved disulfide bonds.

[0194] Example 3 V NAR Expression, purification and structural property analysis

[0195] 1. Construction of the expression carrier

[0196] An intracellular expression vector for BL21 was constructed, and the IIIa protein was expressed using the BL21 intracellular expression method. The coding region of the IIIa gene was amplified using the primer pair IIIa lite to 28a F / IIIa lite to 28a F and ligated between the restriction endonuclease sites BamHI and XhoI of the pET28a expression vector.

[0197] WK6 periodic expression vector, V NAR Protein expression was performed using the WK6 pericytic expression method. The phage particle pADL23c itself can be used as a WK6 pericytic expression vector, therefore no construction was necessary.

[0198] Construct the Expi293F eukaryotic expression vector, V NAR Protein expression was performed using the Expi293F eukaryotic expression method. Equal amounts of upstream primers VNAR to Fc F1 / VNAR to Fc F2 and downstream primers VNAR to Fc R1 / VNAR to Fc R2 were mixed to amplify the VNAR sequence, which was then ligated between the restriction endonuclease sites BamHI and EcoRI of the pcDNA3.4-hIgG-Fc expression vector.

[0199] 2. Periplasmic expression of Escherichia coli strain WK6

[0200] Take the expression of nanobody sequences (His-tags) on phage particles using 300 mL of bacterial culture as an example.

[0201] (1) A single clone of the WK6 strain transformed with phage particles was inoculated into 5 mL of 2×YT culture medium and cultured overnight on a shaker (37℃, 200 rpm) to prepare the bacterial strain. The strain was then transferred to 300 mL of TB culture medium and cultured on a shaker until OD... 600 =0.5 (37℃, 200rpm), add IPTG to a final concentration of 1mM, and incubate in a constant temperature shaker (25℃, 16h, 200rpm).

[0202] (2) Centrifuge to settle the bacterial cells (5000×g, 5min, 4℃), discard the supernatant, and place on ice. Resuspend in 25mL of TES solution pre-cooled at 4℃, incubate on a shaker (4℃, 1h, 120rpm), then add 50mL of TES / 4 solution pre-cooled at 4℃, and incubate on a shaker (4℃, 45min, 120rpm).

[0203] (3) Centrifuge to settle bacterial cell fragments (12000×g, 30min, 4℃), and retain a small amount of precipitate as a sample. Collect the supernatant, add Ni-NTA resin for nickel column purification, and follow the same steps as the His-tagged protein BL21 expression purification method described above.

[0204] 3. Secretory expression of human cell line Expi293F

[0205] Plasmids were extracted using the M5Multi Color Endo Free Plasmid Midi Kit for cell transfection (toxin removal).

[0206] Proteins with Fc tags were expressed using the pcDNA3.4-hIgG Fc vector.

[0207] (1) Resuscitation of Expi293F cell line

[0208] ① Remove the cryovial containing Expi293F cells from liquid nitrogen and thaw it rapidly in a 37°C water bath.

[0209] ② Centrifuge to settle the cells (300×g, 5 min), discard the supernatant, resuspend the cells in a small amount of Union 293 complete culture medium, seed into 30 mL of Union 293 complete culture medium, and incubate on a CO2 shaker (37℃, 8% CO2, 135 rpm) until the cell density reaches 3.0×10⁶ cells / mL. 6 Once cells / mL are reached, passage can begin.

[0210] (2) Passaging of the Expi293F cell line

[0211] ① Seed the revived Expi293F cells into 25 mL of fresh Union 293 complete culture medium per flask to achieve an initial cell density of 0.5 × 10⁻⁶ cells / mL. 6 cells / mL.

[0212] ② Culture in a carbon dioxide constant temperature shaker (37℃, 8% CO2, 135 rpm) until the cell density reaches 5.0 × 10⁻⁶ cells / year. 6 If the cell / mL ratio is high, then passage is required.

[0213] (3) Transfection of Expi293F cell line

[0214] ① One day before transfection, passaged cells to an initial cell density of 1.0 × 10⁶ cells / year. 6 cells / mL, cultured in a carbon dioxide constant temperature shaker (37℃, 8% CO2, 24h, 135rpm).

[0215] ② On the day of transfection, the cell density reached 2.0 × 10⁶ cells / year. 6 Cells / mL, centrifuge to settle cells (100×g, 5min), discard supernatant, resuspend cells in 50mL of fresh Union 293 complete culture medium, and culture in a carbon dioxide constant temperature shaker (37℃, 8% CO2, 1h, 135rpm).

[0216] ③ Dilute 150 μg of plasmid to 300 μL with Opti-Mem serum-free culture medium, add to the cell culture medium, and mix well. Dilute 1 μg / μL of PEI solution to 900 μL with Opti-Mem serum-free culture medium, add to the cell culture medium, mix well, and incubate on a CO2 constant temperature shaker (37℃, 8% CO2, 24h, 135rpm).

[0217] ④ Add VPA to 50 mL of fresh Union 293 complete culture medium to a final concentration of 4.4 mM, mix well, and then add to the cell culture medium.

[0218] ⑤ Starting from the day VPA is added, culture in a constant temperature shaker with carbon dioxide (37℃, 8% CO2, 5 days, 135 rpm), and collect the cell slurry in culture for protein purification.

[0219] (4) Purification of Fc-tagged secretory protein

[0220] ① Take Protein A resin, mix well, take 1 mL, add it to the protein purification column, and let it pass through the column naturally to remove the protective solution.

[0221] ② Centrifuge the cells (2000×g, 10 min, 4℃) and collect the supernatant. Filter the supernatant through a 0.45 μm pore size filter membrane, dilute it 1:1 with Binding Buffer, resuspend the Protein A resin in the protein purification column and add it to the supernatant. Incubate on a shaker (4℃, 1 h, 120 rpm) to allow the Fc-tag protein to bind to the Protein A resin.

[0222] ③ Pass the cell supernatant containing Protein A resin through a protein purification column and rinse for impurities with 6 column volumes of Binding Buffer.

[0223] ④ Stopper the bottom of the purification column, transfer 1 mL of Elution Buffer into the column, and use a rotary mixer to elute the protein (2 min, 4℃). Collect the eluent. Add 1:10 volume of Neutralization Buffer to the eluent to neutralize the pH, thus obtaining the purified sample of the Fc-tagged protein.

[0224] 4. Electrophoresis detection of protein samples

[0225] ① Add 5×SDS loading buffer to the protein purification elution sample, mix thoroughly, and boil in a water bath for 10 minutes to mix thoroughly.

[0226] ② Load the sample into the loading well of the SDS-PAGE gel, use a vertical electrophoresis tank, electrophoresis at 80V for 30 min, then at 120V for 1.5 h.

[0227] ③ Remove the gel, immerse it in Coomassie brilliant blue staining solution, place it on a decolorizing shaker, and stain at room temperature for 2 hours.

[0228] ④ Recover the dye solution, immerse the gel in Coomassie Brilliant Blue decolorizing solution, place it on a decolorizing shaker, and decolorize overnight at room temperature.

[0229] ⑤ Use a gel imaging system to record protein bands on the gel.

[0230] Among them, strain WK6 expresses V in the prokaryotic periplasm. NAR like Figure 21 As shown.

[0231] Expi293F human suspension cell line expresses V NAR like Figure 22 As shown, Expi293F eukaryotic expression V NAR High yield, except for Pirin-V resistance NAR Except for G9, all other sequences were successfully expressed.

[0232] 5. Analysis of the physicochemical properties of antibody sequences

[0233] Calculate V using the Expasy-ProtParam tool NAR The physicochemical properties. The above 11 V NAR The physicochemical properties of all V are shown in Table 15. NAR The molecular weights of all sequences are around 12 kDa. Of the 11 sequences, 8 have isoelectric points higher than the physiological pH (7.35-7.45), indicating a positive charge under physiological conditions, while the remaining 3 sequences carry a negative charge under physiological conditions. The average total hydrophilicity coefficient of all sequences is negative, indicating that V... NAR As the protein is hydrophilic, the above results are in line with expectations.

[0234] Table 15 V NAR Physicochemical properties of the sequence

[0235]

[0236]

[0237] Note: MW represents molecular weight. pI represents isoelectric point. GRAVY represents the average coefficient of total hydrophilicity; GRAVY < -0.5 indicates high hydrophilicity.

[0238] Example 4V NAR Affinity Measurement and Verification

[0239] 1. BLI assay for antigen-antibody affinity

[0240] (1) Determine the protein sample concentration using the BCA method.

[0241] ① Prepare BSA concentration gradients and samples. First, prepare a 2 μg / μL BSA-PBS solution, and repeatedly halve it to obtain 9 concentration gradients, down to 1 / 128 μg / μL. Add the BSA concentration gradient sequentially to a 96-well microplate, 10 μL per well, with three parallel wells for each concentration. Add the sample to the 96-well microplate, 10 μL per well, with three parallel wells for each concentration.

[0242] ② Colorimetric reaction. Mix BCA stock solution A and solution B thoroughly to prepare the working solution. Add 90 μL of the working solution to each BSA / sample well, incubate at 37°C for 30 min, take photos to record the results, and measure the absorbance (OD) using a microplate reader. 562 .

[0243] ③ BSA concentration gradient standard curve fitting and concentration determination. The average value of all parallel wells for all samples is taken. A linear function curve is fitted with the absorbance corresponding to the BSA concentration gradient as the independent variable and the BSA concentration as the dependent variable. The sample absorbance is then substituted into the independent variable to obtain the sample concentration.

[0244] (2) BLI Sample Preparation and Procedure Setup. Each row represents one sample. From the first to the fifth column, PBS TV is added to each sample. NAR The sample-PBST-antigen sample-PBST sequence corresponds to the Baseline 1-Loading-Baseline 2-Association-Dissociation steps in the BLI assay. For each antigen-antibody pair, five antigen concentration gradients were designed, plus a blank control group with an antigen concentration of 0. The specific procedure is shown in Table 16.

[0245] Table 16 BLI Affinity Measurement Procedure

[0246]

[0247] For Fc-tagged proteins, Protein A probes were used, and for biotinylated proteins, SA probes were used. After using Protein A probes, the following procedure was designed to recover the probes (Table 17).

[0248] Table 17 Protein A Probe Recovery Procedure

[0249]

[0250]

[0251] The results are as follows Figures 23-27 As shown, V expressed in the periplasm of E. coli was first used. NAR BLI testing was performed, but antigen-V NAR The combined signal is generally weak, and V NAR The measured values ​​of the equilibrium dissociation constant were generally too low. Subsequently, V expressed in the Expi293F suspension cell line was used. NAR BLI measurements were repeated. In the 16 V samples that participated in the measurement... NAR The clip contains 11 V's. NAR The fragment exhibits high affinity and corresponds to five antigens. The binding curves obtained from the affinity assay are shown below. Figures 23-27As shown in Table 18, the equilibrium dissociation constant, binding rate, and dissociation rate of each antigen-antibody pair are as follows.

[0252] Table 18 Affinity constants of high-affinity antibodies

[0253]

[0254] Note: Response represents the signal intensity of antigen-antibody binding (unit: nm). A signal that is too low is not conducive to accurate measurement; a higher signal is better. K D k is the equilibrium dissociation constant; a lower value indicates higher affinity. a This refers to the antibody binding rate; generally, the higher the better. d This represents the antibody dissociation rate, and generally, a lower rate is better. The data in the table are the average of all concentration gradient measurements, with the nearest 0.5 taken.

[0255] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. The illustrative expressions of the above terms in this specification should not be construed as necessarily referring to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. In addition, those skilled in the art can combine and integrate the different embodiments or examples described in this specification.

[0256] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A nanobody targeting Pirin, characterized in that, The nanobody includes a complementarity-determining region (CDR) of: CDR1 shown in SEQ ID NO: 1 and CDR3 shown in SEQ ID NO: 2, or CDR1 shown in SEQ ID NO: 10 and CDR3 shown in SEQ ID NO: 11, and an amino acid sequence of HV2 shown in SEQ ID NO: 3 and an amino acid sequence of HV4 shown in SEQ ID NO: 4, or HV2 shown in SEQ ID NO: 12 and HV4 shown in SEQ ID NO:

13.

2. The pirin-targeting nanobody as described in claim 1, characterized in that, The framework region of the nanobody includes FR1, FR2, FR3 and FR4, the amino acid sequence of FR1 is shown in SEQ ID NO: 5 or SEQ ID NO: 14, the amino acid sequence of FR2 is shown in SEQ ID NO: 6 or SEQ ID NO: 15, the amino acid sequence of FR3 is shown in SEQ ID NO: 7 or SEQ ID NO: 16, and the amino acid sequence of FR4 is shown in SEQ ID NO: 8 or SEQ ID NO:

17.

3. The pirin-targeting nanobody as described in claim 1, characterized in that, The nanobodies include nanobodies with amino acid sequences as shown in SEQ ID NO: 9 or SEQ ID NO:

18.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes a nanobody targeting Pirin as described in any one of claims 1-3.

5. A construct, characterized in that, It includes the nucleic acid molecule as described in claim 4.

6. The use of the pirin-targeting nanobody as described in claim 1 in the preparation of antitumor drugs.

7. The application of the pirin-targeting nanobody as described in claim 1 in the preparation of tumor diagnostic / prognostic reagents, or in the preparation of diagnostic / prognostic kits.

8. The application of the pirin-targeting nanobody as described in claim 1 in the preparation of tumor immunosensitizers.

9. A pharmaceutical composition, characterized in that, Including the pirin-targeting nanobody as described in claim 1.

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