Anti-CD13 Raja sinensis nano antibody and application thereof

Abstract

The invention provides an anti-CD13 Raja sinensis nano antibody and application thereof. In a complementary determining region of the antibody, the amino acid sequence of CDR1 is shown as SEQ ID NO: 1, and the amino acid sequence of CDR3 is shown as SEQ ID NO: 2 or SEQ ID NO: 3. The antibody is soluble in water, has activity and can be used for BLI determination of antigen-antibody affinity, the development period and cost of the antibody are remarkably reduced, and the superiority of the Chinese rhodiola rosea source VNAR sequence in the aspect of hydrophilicity is indicated; the KD value of the CD13 antigen and one antibody is measured to be 159.3 nM and is at the medium level of the affinity of the natural VNAR antibody; a molecular tool is provided for developing a novel CD13 inhibitor, and the clinical transformation problem of the inhibitor is expected to be solved.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to an anti-CD13 nanobody of the Chinese fan ray and its application. Background Technology

[0002] Cartilaginous fish IgNARs do not contain heavy chain antibodies, and VNARs possess broad antigen recognition and binding capabilities, making them an important research subject. However, current research on cartilaginous fish VNARs primarily focuses on sharks, while rays (such as the Chinese fan ray) are less studied. Platyrhina sinensis Research on this topic is still relatively rare.

[0003] CD13 (also known as aminopeptidase N) is a hippocampal-shaped enzyme that forms head-to-head homodimers through hydrophobic interactions. Each monomer consists of seven domains and is a widely expressed type II metalloproteinase. CD13 possesses not only enzymatic activity (cleaving the N-terminus of cytokines, hormones, and MHC II-binding peptides, thereby regulating their activity) but also non-enzymatic activity (signal transduction, receptor interaction, phagocytosis regulation, etc.). Due to its high expression in various tumor cells and its significant association with poor prognosis (reduced survival rate, increased risk of metastasis), CD13 has long been used as a biomarker for characterizing and classifying leukemia and lymphoma cells, and may be a potential therapeutic target for inflammatory diseases. As an important cell surface marker, CD13 also plays a crucial role in regulating the development and function of immune-related immune cells; and it plays an important role in the pathogenesis of many inflammatory diseases (rheumatoid arthritis, psoriasis, multiple sclerosis, inflammatory bowel disease, osteoarthritis, etc.). Studies have found that CD13 is closely related to the pathogenesis of many inflammatory diseases and is an important therapeutic target. Although CD13 inhibitors have anti-inflammatory effects, clinical translation is still in its infancy, and treatment strategies targeting CD13 are urgently needed. Summary of the Invention

[0004] To address the aforementioned issues, this invention proposes an anti-CD13 nanobody of the Chinese fan ray and its application, which can precisely bind to the CD13 antigen, providing a molecular tool for the development of new CD13 inhibitors and potentially solving the clinical translation problem of such inhibitors.

[0005] To achieve the above objectives, embodiments of the present invention provide a first aspect of an anti-CD13 Chinese fan ray nanobody, wherein the amino acid sequence of CDR1 in the complementarity-determining region of the antibody is shown in SEQ ID NO:1, and the amino acid sequence of CDR3 is shown in SEQ ID NO:2 or SEQ ID NO:3.

[0006] According to an embodiment of the present invention, a CD13-based nanobody of the Chinese fan ray is derived from total RNA isolated from spleen tissue cells of the Chinese fan ray. This RNA was reverse transcribed into cDNA, and the VNAR sequence was amplified using specific primers. Subsequently, a natural VNAR phage display library of the Chinese fan ray was constructed, and the library was screened using CD13 protein as an antigen, ultimately successfully identifying the desired sequence. Prokaryotic and eukaryotic expression of the CD13-specific VNAR sequence were performed, revealing its water solubility and activity, allowing for BLI assays to determine antigen-antibody affinity. This significantly reduces the antibody development cycle and cost, demonstrating the superior hydrophilicity of the Chinese fan ray-derived VNAR sequence. The Kg of the CD13 antigen and one of the antibodies was measured. D With a value of 159.3 nM, it is at a moderate level of affinity for natural VNAR antibodies; it provides a molecular tool for the development of new CD13 inhibitors and is expected to solve the problem of clinical translation of such inhibitors.

[0007] Optionally, the frame region of the antibody includes FR1, FR2, FR3a, FR3b and FR4, wherein the amino acid sequence of FR1 is shown in any of SEQ ID NO: 6-9, the amino acid sequence of FR2 is shown in SEQ ID NO: 10, the amino acid sequence of FR3a is shown in SEQ ID NO: 11, the amino acid sequence of FR3b is shown in SEQ ID NO: 12 and the amino acid sequence of FR4 is shown in SEQ ID NO: 13.

[0008] Optionally, the hypervariable ring of the antibody includes HV2 and HV4, the amino acid sequence of HV2 is shown in SEQ ID NO:4, and the amino acid sequence of HV4 is shown in SEQ ID NO:5.

[0009] Optionally, the antibody comprises a nanobody with an amino acid sequence as shown in any of SEQ ID NO:14-17.

[0010] 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-mentioned anti-CD13 Chinese fan ray nanobody.

[0011] According to an embodiment of the present invention, a third aspect provides a construct comprising the above-described nucleic acid molecule.

[0012] According to an embodiment of the present invention, in a fourth aspect, the above-described Chinese fan ray nanobody is proposed for use in the preparation of CD13 inhibitors.

[0013] According to an embodiment of the present invention, a pharmaceutical composition comprising the above-described anti-CD13 Chinese fan ray nanobody is provided in a fifth aspect.

[0014] 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

[0015] Figure 1 This is a gel electrophoresis image of VNAR PCR amplification in Example 1 of the present invention. M is the marker, and the other lanes are the PCR results of the VNAR target fragment. Figure 2 This is a pADL-23C VNAR phage particle map of an embodiment of the present invention; Figure 3 This is a plasmid map of pcDNA3.4-hIgGFc-CD13 from an embodiment of the present invention; Figure 4 This is an SDS-PAGE electrophoresis image of CD13 protein according to an embodiment of the present invention. M is the marker, and lane 1 is the CD13 protein. Figure 5 This describes the CD13 enrichment situation in an embodiment of the present invention. Figure 6 This is the phage ELISA of the present invention for detecting the binding ability of 96 VNARs to CD13. The red dashed line is the boundary between positive and negative colonies. Figure 7 This is a differential analysis of four anti-CD13 VNAR amino acid sequences in an embodiment of the present invention; Figure 8 These are SDS-PAGE electrophoresis images of prokaryotic and eukaryotic anti-CD13 VNAR expressed in an embodiment of the present invention. A: Protein expressed in the prokaryotic expression system, lane 1 is A2, lane 2 is C2, lane 3 is C9, and lane 4 is E12; B: Protein expressed in the eukaryotic expression system, lane 1 is Fc-A2, and lane 2 is Fc-C9. Figure 9 This is the result of the dynamic analysis of the interaction between CD13 and C9-Fc in an embodiment of the present invention; Figure 10 This is a CD13-C9 molecular docking prediction model according to an embodiment of the present invention; the yellow part is the CD13 antigen, and the pink part is the C9 antibody. Detailed Implementation

[0016] 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.

[0017] 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.

[0018] Culture medium and reagent formulations: 2×YT medium: peptone 16 g / L, yeast extract 10 g / L, NaCl 5 g / L. For solid medium, add agar 15 g / L. Autoclave at 121℃ for 20 min, and add antibiotics after cooling to below 60℃.

[0019] TB medium: peptone 12 g / L, yeast extract 24 g / L, 2% glycerol (volume). Autoclave at 121°C for 20 min, and add antibiotics after cooling to below 60°C. Before use, supplement with 0.1% filtered sterile (0.22 μm) 50% glucose and 1M MgSO4.

[0020] LB medium: peptone 16 g / L, yeast extract 10 g / L, NaCl 5 g / L; for solid media, add agar 15 g / L. Autoclave at 121°C for 20 min. Cool to below 60°C, antibiotics can be added.

[0021] PBS buffer: Add PBS phosphate buffer (dry powder) BL601A to ultrapure water and bring the volume to 2 L.

[0022] 0.1% PBST buffer: Add 1 mL of Tween-20 to 1 L of PBS buffer, mix well, and store at room temperature.

[0023] 0.02% PBST buffer: Add 200 μL Tween-20 to 1 L PBS buffer, mix well, and filter through a 0.22 μm filter for use in subsequent BLI experiments. Prepare fresh before use.

[0024] PEG6000: Dissolve 200 g of PEG6000 and 146.11 g of NaCl in ultrapure water, and bring the volume to 1 L. Autoclave at 121°C for 20 min. After cooling to below 50°C, mix the sterilized layered liquids with a magnetic stirrer until a homogeneous consistency is achieved, and store at room temperature.

[0025] Ampicillin stock solution (100 mg / mL): Dissolve 2 g of ampicillin in ultrapure water and bring the volume to 20 mL. Filter through a 0.22 μm filter, aliquot, and store at -20°C.

[0026] Kanamycin sulfate stock solution (50 mg / mL): Dissolve 1 g of kanamycin sulfate in ultrapure water and bring the volume to 20 mL. Filter through a 0.22 μm filter, dispense, and store at -20°C.

[0027] TB buffer: PIPES 10 mM, CaCl2 15 mM, KCl 250 mM, MnCl2 55 mM. After dissolving PIPES (PIPES is insoluble in water, a suitable amount of 0.5 M KOH can be added to aid dissolution), add CaCl2 and KCl, adjust the pH to 6.7 with 0.5 M KOH, and finally add MnCl2. After complete dissolution, filter through a 0.22 μm filter into sterile blue-neck bottles and store at 4°C.

[0028] Cell culture medium: Before use, add 1% by volume of 50% glucose and GlutaMAX-I (100×) to Union 293 medium, mix well and store at 4℃.

[0029] Bind Buffer: NaCl 0.15M, Na2HPO4 20mM. Dissolve the above components in ultrapure water, mix well, adjust the pH to 7.0, filter through a 0.45 μm filter, and store at 4℃.

[0030] Elution Buffer: Adjust pH to 3.0 with 0.1 M glycine, filter through a 0.45 μm filter, and store at 4°C.

[0031] Neutralization Buffer: Adjust pH to 8.5 with 0.1 M Tris-HCl, filter through a 0.45 μm filter, and store at 4°C.

[0032] Separating gel solution (5 mL) 10% separating gel: 1.667 mL of 30% acrylamide, 1.25 mL of 4×Tris-HCl-SDS (pH 8.8), 2.083 mL of H2O, 0.017 mL of 10% APS, and 0.003 mL of TEMED. Mix all components thoroughly before use. Prepare and use immediately.

[0033] 12% separating gel: 2 mL of 30% acrylamide, 1.25 mL of 4×Tris-HCl-SDS (pH 8.8), 1.75 mL of H2O, 0.017 mL of 10% APS, and 0.003 mL of TEMED. Mix all components thoroughly before use. Prepare and use immediately.

[0034] 15% separating gel: 2.5 mL of 30% acrylamide, 1.25 mL of 4×Tris-HCl-SDS (pH 8.8), 1.25 mL of H2O, 0.017 mL of 10% APS, and 0.003 mL of TEMED. Mix all components thoroughly before use. Prepare and use immediately.

[0035] 3.9% Stacking Gel Solution (5 mL): 0.65 mL of 30% Acrylamide, 1.25 mL of 4×Tris-HCl / SDS buffer (pH 6.8), 3.1 mL of H2O, 0.025 mL of 10% APS, and 0.005 mL of TEMED. Mix all components thoroughly before use. Prepare fresh before use.

[0036] 5×SDS PAGE protein electrophoresis buffer: Glycine 72 g / L, Tris 15.1 g / L, and SDS 5 g / L dissolved in ultrapure water to prepare the stock solution. The working solution needs to be diluted to 1× before use.

[0037] 5×SDS protein loading buffer: 25 mL 1 M Tris-HCl (pH 6.8), 10 g SDS, 0.5 g bromophenol blue, 41.67 mL glycerol, 10 mL 5 M DTT. Add ultrapure water to a final volume of 100 mL, aliquot, and store at -20°C.

[0038] Coomassie Brilliant Blue Staining Solution Methanol:acetic acid:ultrapure water = 4:1:5 (volume ratio), plus 2.5 g / L Coomassie Brilliant Blue R250, mix well and store at room temperature.

[0039] Coomassie Brilliant Blue Decolorizing Solution Methanol:acetic acid:ultrapure water = 4:1:5 (volume ratio), mix well and store at room temperature.

[0040] Buffer A: Tris 25mM, NaCl 150mM, imidazole 20mM, 5% glycerol, adjust pH to 8.0, filter through a 0.45 μm filter membrane and store at 4°C.

[0041] Buffer B: Tris 25mM, NaCl 150mM, imidazole 500mM, 5% glycerol, adjust pH to 8.0, filter through a 0.45 μm filter membrane and store at 4°C.

[0042] 100 mM PMSF: Dissolve 0.174 g of PMSF powder in 10 mL of isopropanol, dispense, and store at -20°C.

[0043] IPTG (500 mM): Dissolve 12 g of IPTG powder in 100 mL of ultrapure water, filter through a 0.22 μm filter, dispense, and store at -20°C.

[0044] Biotin-NHS stock solution: Add 100 mg of Biotin-NHS to 10 mL of DMSO, aliquot, and store at -20°C.

[0045] Primer names and sequences used in the examples:

[0046] 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.

[0047] Example 1: Construction of a nanobody phage library of the Chinese fan ray Chinese fan ray samples were purchased from the Eighth Seafood Market in Xiamen, Fujian Province. Fresh individuals with strong activity were selected. After pretreatment with MS-222 anesthesia, the spleen tissue was obtained through live dissection, quickly placed in pre-cryogenic storage tubes, flash-frozen in liquid nitrogen for 1 minute, and then stored in liquid nitrogen at -80℃ for long-term preservation.

[0048] 1. Total RNA was extracted from spleen tissue using the TRIzol method. (1) Take spleen tissue from Chinese fan ray, add 3 mL of TRIPure reagent to every 50 mg, cut into small pieces and homogenize with a homogenizer until the texture is uniform. The tissue homogenate can be temporarily stored at -80℃ or enter the extraction process. (2) Add 200 μL of chloroform to each 1 mL of tissue homogenate, shake vigorously for 30 s to mix, let stand at room temperature for 10 min, then centrifuge at 15000×g for 10 min at 4℃, and transfer the upper aqueous phase (about 500 μL) to a new EP tube. (3) Add an equal volume (500 μL) of pre-cooled isopropanol, gently invert and mix 10 times, let stand at room temperature for 10 min, centrifuge at 15000×g for 10 min at 4℃, then discard the supernatant, the white precipitate is crude RNA. (4) Add 1 mL of ethanol to the precipitate, centrifuge at 15000×g for 5 min at 4℃, discard the supernatant and dry at room temperature for 6 min (avoid over-drying).

[0049] 2. Total RNA was reverse transcribed into cDNA Total RNA from spleen tissue was reverse transcribed into complementary DNA (cDNA) using the ABScript II cDNA First-Strand Synthesis Kit. The mixed reverse transcription reaction system is shown in Table 1 below: Table 1. RNA reverse transcription reaction system (20 μL)

[0050] After the above reaction system was thoroughly mixed, it was placed in a PCR instrument for reverse transcription. The reaction program was set as follows: 42℃, 60 min; 80℃, 5 min. After the reaction, the cDNA product was cooled on ice and then stored at -20℃.

[0051] 3. cDNA purification using magnetic beads (1) Wash the cDNA with Hieff NGS® DNA Selection Beads. Before use, the DNA selection beads should be equilibrated at room temperature for at least 30 minutes to avoid affecting the binding efficiency due to temperature differences.

[0052] (2) Take 350 μL of cDNA product and mix it with an equal volume of magnetic beads. Mix well by pipetting 10 times and incubate at room temperature for 5 min to complete nucleic acid adsorption.

[0053] (3) After a brief centrifugation, place the tube on a magnetic rack for 5 min until the solution becomes clear. At this point, all the magnetic beads will be adsorbed onto the wall of the centrifuge tube. Slowly remove the supernatant. Keep the tube in a fixed position on the magnetic rack, add 875 μL of 80% ethanol to wash the magnetic beads, let stand for 30 s, remove the supernatant, and repeat the washing once. Open the lid and dry at room temperature for 5 min.

[0054] (4) Add 150 μL of ultrapure water and mix by pipetting. Incubate at room temperature for 5 min and then place on a magnetic rack for magnetic separation. After the liquid becomes clear, transfer the supernatant to a clean EP tube. The cDNA purified by magnetic beads can be stored at -20℃.

[0055] 4. Amplification of the VNAR gene Using cDNA purified from magnetic beads as a template, PCR was performed using 2×F8 FastLong PCR MasterMix enzyme to amplify the VNAR target fragment. The reaction system is shown in Table 2, and the VNAR reaction program is shown in Table 3. Table 2 R VNAR amplification reaction system

[0056] Table 3 VNAR amplification reaction procedure

[0057] After the PCR amplification reaction is complete, immediately transfer the sample to ice to cool. Once the temperature has dropped to room temperature, perform electrophoresis. Remove the gel block corresponding to the target band location and purify and recover the VNAR PCR product using a gel extraction kit. Use Nanodrop 2000 to determine the concentration of the VNAR amplification product. The product can be stored at -20℃ for subsequent enzyme digestion reactions. Figure 1 As shown, the amplification product exhibits the target band at a size of approximately 300 bp.

[0058] 5. Construction and transformation of phage display particles Using restriction endonucleases Bgl I. The phage display vector pADL-23C and the VNAR gene amplification product were digested with enzymes to generate sticky ends for subsequent ligation and library construction. The enzyme digestion systems for the vector and the target fragment are shown in Tables 4 and 5, respectively. Table 4. Linearization reaction system of pADL23c carrier

[0059] use Bgl Ⅰ. After restriction endonuclease digestion at 37℃ for 2 h, add 4 μL of quick CIP to the vector digestion system (VNAR digestion does not require this addition), and continue digestion for 1 h. After the reaction, the pADL-23C digested vector is run on a gel and recovered from the gel, while the VNAR digested fragment is recovered from the gel without running on the gel.

[0060] Table 5 VNAR gene digestion system

[0061] The linearized phage display vector pADL-23C and the VNAR gene fragment were ligated using T4 ligase via sticky end ligation, resulting in a complete circular plasmid, as shown in the diagram. Figure 2 As shown, the connection system is as shown in Table 6: Table 6. Linkage system of pADL23c vector and VNAR fragment

[0062] The cells were incubated overnight at 4°C, and then inactivated by water bath at 70°C for 15 min. After that, the cells were recovered without gel running and ready to be transferred into TG1 competent cells.

[0063] TG1 competent state fabrication and electroporation: Prepare in advance: (1) Five bottles, four large and one small (with ridges at the bottom to increase dissolved oxygen), were ultrasonically cleaned and then soaked overnight in 1 M NaOH solution, and then rinsed twenty times with ultrapure water.

[0064] (2) 24.6 μg of VNAR target fragment was digested with enzymes, and 71.4 μg of pADL-23C vector was digested with enzymes. The mass ratio of vector to target fragment was 3:1.

[0065] (3) Streak TG1 plates and incubate overnight at 37°C.

[0066] (4) Prepare 10 mL and 100 mL × 4 TB culture medium in conical flasks that have been soaked and cleaned with NaOH solution. Use Wahaha water to prepare and sterilize the medium. Before use, add 1‰ 50% glucose and 1‰ 1 M MgSO4 respectively. Sterilize a clean 1 L empty conical flask for later use.

[0067] (5) Clean the electroshock cup with a dental irrigator in advance, rinse the 0.1 mm gap of the electroshock cup five times with ultrapure water, add 1 mL of 0.2 M HCl to each electroshock cup and let it stand at room temperature for 10 min, rinse the gap of the electroshock cup five times with 70% alcohol, store it in a clean container wiped with alcohol, and dry it in an oven.

[0068] (6) Sterilize 30 large iron plates (40×40 cm) in advance, and sterilize the scraper in advance.

[0069] (7) 800 mL 2×YT solid culture medium (30 bottles for inverted plates), several 10 cm petri dishes and counting plates.

[0070] (8) Before preparing TG1 competent cells, 10% pre-cooled glycerol needs to be prepared, and the electrocup needs to be pre-cooled.

[0071] Experimental steps: (1) Streaking the frozen TG1 onto antibiotic-free 2×YT medium and incubating overnight at 37°C. Picking single colonies and transferring them to 10 mL of TB medium and incubating overnight at 37°C and 220 rpm on a shaker to obtain TG1 stock solution.

[0072] (2) The TG1 stock solution was inoculated into 100 mL of TB medium at a ratio of 1:100, and cultured at 18℃ and 250 rpm for 21 h. The OD was measured after dilution by 10 times. 600≈0.3.

[0073] (3) Fill each 50 mL centrifuge tube with 25 mL of bacterial culture, centrifuge vertically at 3000×g for 10 min at 4℃, gently discard the supernatant, centrifuge vertically at 3000×g for 1 min at 4℃, and use a pipette to remove the culture medium.

[0074] (4) Add 30 mL of 10% pre-cooled glycerol to each tube, gently resuspend the bacterial cells, centrifuge vertically at 3000×g for 10 min at 4℃, discard the supernatant, centrifuge vertically at 3000×g for 1 min at 4℃, and aspirate the culture medium with a pipette.

[0075] (5) Add 20 mL of 10% pre-cooled glycerol to each tube, gently resuspend the bacterial cells, and rotate vertically on a vertical mixer until the bacterial solution is mixed evenly. Combine the bacterial solutions from 4 tubes into 2 tubes, centrifuge vertically at 3000×g for 10 min at 4℃, discard the supernatant, centrifuge vertically at 3000×g for 1 min at 4℃, and remove the culture medium with a pipette.

[0076] (6) Add 20 mL of 10% pre-cooled glycerol to each tube, gently resuspend the bacterial cells, and vertically rotate the tube on a vertical mixer until the bacterial solution is thoroughly mixed. Combine the two tubes into one tube, centrifuge vertically at 3000×g for 10 min at 4℃, discard the supernatant, and centrifuge vertically at 3000×g for 1 min at 4℃. Use a pipette to remove the culture medium completely. Gently resuspend the bacterial cells, dilute 100 times, and measure the OD. 600 The optimal original OD value is 100-150, and the final volume is approximately 5 mL.

[0077] (7) Add T4 enzyme ligation gel recovery product (about 51 μg) to the electrotransfer competent cells, mix well, place on ice, add 50 μL of bacterial solution to each pre-cooled electrotransfer cup, and electrotransform at 1.25 KV for 4 ms.

[0078] (8) After electroporation, 1 mL of TB medium was added to each electroporation cup of bacterial culture. The culture was resuspended using a syringe (23 mm) and finally transferred to a clean empty conical flask. 93 electroporation cups were electroporated to obtain 200 mL of bacterial culture. After culturing on a shaker at 37°C for 1 h, the culture was spread on 40×40 cm solid plates (ampicillin resistant, 30 plates in total) and incubated overnight at 37°C. At the same time, counting plates were also used to estimate the stock volume.

[0079] (9) The steps for diluting and plating the counting plate are as follows: Take 10 μL of 200 mL of bacterial culture and add it to 990 μL of 2×YT medium (10 4 (Diluted solution) Take 10 μL and apply it as 10 μL. 6 Counting board, at 10 4 Take 10 μL of the diluted solution and add it to 990 μL of 2×YT medium (106 (Diluted solution) Take 100 μL and spread it to obtain 10. 7 For the counting plate, take 10 μL and spread it to obtain 10. 8 For the counting plate, take 1 μL and spread it to obtain 10. 9 Counting board.

[0080] 6. Phage library amplification and purification (1) Cell preservation. TG1 Escherichia coli cultured overnight on 40×40 cm solid plates (containing 100 μg / mL ampicillin) was collected using a sterile scraper, mixed with 20% glycerol, and aliquoted into 50 mL centrifuge tubes (40 mL per tube) for long-term storage at -80℃. A single tube of bacterial culture was used for subsequent phage library preparation.

[0081] (2) Cell amplification. 40 mL of electroporated TG1 bacterial culture was evenly inoculated into 12 cells of 500 mL 2×YT medium (or initial OD). 600 =0.1), add 500 μL of ampicillin (100 mg / mL) to each bottle, incubate at 37℃ with shaking at 220 rpm for 2 h until OD. 600 =0.5.

[0082] (3) Facilitate phage infection. Add 500 μL of laboratory-prepared helper phage M13KO7 (≥1×10⁻⁶) to each culture medium. 13 Infected at 37°C and 220 rpm for 1 hour with PFU / mL incubation.

[0083] (4) Secondary amplification. Add 500 μL of kanamycin (30 mg / mL) to each culture medium, change the incubator temperature to 25℃, and incubate overnight with shaking at 220 rpm.

[0084] (5) Remove bacterial cells. Centrifuge the culture medium at 9000×g for 20 min at 4℃ and collect the supernatant into a clean sterile container.

[0085] (6) PEG precipitation. Add 20% of the supernatant volume of PEG6000, place on a horizontal shaker at 4℃ and shake (180 rpm) for 1 h, then centrifuge at 10000×g for 30 min at 4℃ to collect the precipitate.

[0086] (7) Washing and concentration. Resuspend the precipitate in 15 mL PBS, centrifuge at 15000×g for 10 min at 4℃, and collect the supernatant as purified phage (repeat centrifugation twice).

[0087] (8) Titer determination and preservation. OD was determined by diluting the sample 10-fold with PBS. 268 Value (number of bacteriophages = OD) 268 ×5×1012 (PFU / mL), the prepared phage library can be stored at -80℃ for subsequent screening.

[0088] Example 2 1. Construction of recombinant CD13 antigen expression vector The strain used for constructing the protein expression vector was DH5α competent cells, and the vector used in the experiment was pcDNA3.4-hIgG1Fc. This vector was constructed using... EcoR I and BamH The DNA sequence of the Fc fragment of human IgG1 was ligated to the pcDNA3.4 vector (purchased from Baosai Biotechnology) at the I restriction site. The construction of the CD13 antigen protein expression vector required cloning the CD13 sequence into the pcDNA3.4-hIgG1Fc vector, which is 6723 bp in length and ampicillin resistant.

[0089] The antigen CD13 gene sequence (GeneBank: NM_001150.2) was obtained from the NCBI database. After discarding its signal peptide and transmembrane region, the remaining 2697 bp nucleotide sequence is shown in SEQ ID NO: 18.

[0090] use EcoR I and BamH I. The pcDNA3.4-hIgG1Fc vector plasmid was linearized by double restriction endonuclease digestion. The reaction system is shown in Table 7. After mixing the above components, the mixture was incubated at 37°C for 3 h to complete the double restriction digestion reaction. Subsequently, the reaction was detected by agarose gel electrophoresis (120 V, 30 min). The gel block at the target band location was removed, and the linearized vector product was recovered using the SteadyPure DNA Gel Recovery Kit. The product can be stored at -20°C for subsequent ligation reactions.

[0091] Table 7 Linearization reaction system of pcDNA3.4-hIgG1Fc vector

[0092] CD13 target gene amplification: The CD13 template was amplified by PCR according to the following reaction system (50 μL) to obtain the CD13 target fragment. The reaction system is shown in Table 8.

[0093] Table 8 CD13 Amplification PCR Reaction System

[0094] The PCR reaction procedure is shown in Table 9. After the PCR amplification reaction is completed, immediately place the gel on ice to cool. After the temperature drops to room temperature, perform electrophoresis and run the gel. Remove the gel block of the desired size and use a gel extraction kit to purify and recover the product. The product can be stored at -20℃ for subsequent ligation reactions.

[0095] Table 9. CD13 Amplification PCR Reaction Procedure

[0096] Ligation of the target gene with the vector: The linearized pcDNA3.4-hIgG1Fc vector and the CD13 amplification product were ligated using the T5 exonuclease-mediated TEDA method. The linearized vector (enzyme digestion product) and the insert (PCR product) were mixed at a 1:4 molar ratio (total volume 16 μL, with any shortfall made up with ddH2O), and 4 μL of 5×TEDA concentrate (containing 0.04 U T5 enzyme) was added and mixed thoroughly (reaction system shown in Table 10). The mixture was then placed in a PCR amplification instrument at 30℃ for 40 min, successfully constructing the pcDNA3.4-hIgGFc-CD13 recombinant expression vector. The plasmid map is shown below. Figure 3 As shown, after the procedure, the product was cooled on ice and then transferred into E. coli.

[0097] Table 10 Ligation system of pcDNA3.4-hIgG1Fc vector and CD13 fragment

[0098] Linkage product transformation: (1) Take DH5α competent cells stored at -80℃ and thaw them on ice for 5-10 min until they are liquid. Add all the ligation product (20 μL), gently pipette to mix, and incubate on ice for 30-40 min.

[0099] (2) Place the mixture in a 42°C water bath for 90 s heat shock, and immediately transfer it to ice for 5 min incubation.

[0100] (3) Add 1 mL of 2×YT liquid culture medium to the mixture and shake in a shaker (220 rpm) at 37℃ for 40-60 min.

[0101] (4) Centrifuge at 5000×g for 2 min at room temperature to collect the bacterial cells. Discard 1 mL of supernatant and retain 100 μL of solution to resuspend the bacterial cells. Mix the bacterial solution by pipetting and spread it evenly on a 2×YT solid plate containing 100 μg / mL ampicillin. Incubate overnight at 37℃.

[0102] Identification of recombinant plasmids: Table 11 Colony PCR Reaction System

[0103] Five single-clone colonies from the ligation plate were randomly selected and inoculated into 1 mL of LB liquid medium containing ampicillin. The culture was carried out at 37°C with shaking (220 rpm) for 5-6 h. 1 μL of the bacterial culture was used as a template to prepare a 10 μL colony PCR reaction system according to Table 11. The amplification program was set according to Table 12. The reaction was carried out in a PCR instrument.

[0104] Table 12 Colony PCR Reaction Procedure

[0105] After the reaction was completed, the size of the target band was finally detected by agarose gel electrophoresis. Clones containing the expected size band were selected, and recombinant plasmids were obtained using a plasmid extraction kit and sent to Xiamen Platinum Biotech Co., Ltd. for sequencing. Sequence alignment using Snapgene software confirmed the correct insertion of the target gene. Correctly ligated plasmids were stored at -20℃ for later use.

[0106] 2. Eukaryotic expression and purification of recombinant CD13 antigen protein HEK-293F suspension cell culture: (1) Cell resuscitation The cryovials containing HEK-293F suspension cells were rapidly immersed in liquid nitrogen into a 37°C water bath and gently shaken continuously until completely thawed (<2 min). Centrifuged at 300×g for 5 min at room temperature. The supernatant was discarded, and the cells were resuspended in a small amount of Union 293 cell culture medium (with a final concentration of 1% GlutaMAX and a final concentration of 0.5% glucose, the same below), and seeded into 250 mL cell culture flasks containing 30 mL of Union 293 culture medium. The cells were cultured in a CO2 incubator for approximately 72 h (37°C, 8% CO2, with a horizontal shaker at 135 rpm).

[0107] (2) Subculture When the cell density reaches 3-4 × 10 6 At a concentration of cells / mL, collect cells by centrifugation at 500×g for 5 min at room temperature. Discard the supernatant and resuspend the pellet in a small amount of fresh Union 293 medium, using 1×10⁻⁶ cells / mL. 6 Cells / mL were seeded to a final density in 50 mL Union293 medium. Culture conditions were maintained the same as described in the CO2 incubator for subsequent Fc-tagged protein expression.

[0108] Cell transfection and recombinant protein expression: (1) Transfection preparation Cells were passaged one day before transfection at a concentration of 1×10⁻⁶ cells / mL.6 The cells / mL were seeded at a final density and cultured in 50 mL Union293 medium for 24 h.

[0109] (2) Transfection operation On the day of transfection, cells were collected by centrifugation at 500×g for 5 min at room temperature, and then cultured in 50 mL of fresh Union 293 medium for another 1 h. 150 μg of plasmid was diluted with low-serum medium (Opti-MEM) to a final concentration of 0.5 μg / μL, with a total volume of 300 μL. 450 μL of PEI (1 mg / mL) was mixed with an equal volume of low-serum medium to prepare a 0.5 mg / mL PEI solution (polyethyleneimine). After thoroughly mixing the above solutions, the plasmid solution was added to the cell culture medium first, and the mixture was shaken to mix. Then, the PEI solution was added, and the mixture was shaken to mix again. The cells were then incubated in a CO2 incubator for 24 h with shaking.

[0110] (3) Protein-induced expression 24 h after transfection, VPA (valproic acid) solution (220 mM, 1 mL) was diluted to 4.4 mM with 50 mL of culture medium and added to the transfected cell culture medium for further culture for 72-96 h.

[0111] Recombinant protein purification: The recombinant protein constructed based on the pcDNA3.4-hIgGFc eukaryotic expression vector contains an Fc tag at its C-terminus. Utilizing the high specific affinity of Protein A Resin for the Fc tag, a three-step affinity chromatography process (binding-washing-elution) enables efficient capture and purification of the target recombinant protein. The specific purification procedure is as follows: (1) Cell culture medium, 800×g, 20 min, 4℃ centrifugation, the supernatant was filtered through a 0.45 μm filter membrane and mixed with an equal volume of Bind Buffer in a clean cell shake flask.

[0112] (2) Transfer 0.5 mL of Protein A Resin to the chromatography column, rinse and equilibrate with 20 mL of Bind Buffer, add the cell supernatant to the filtrate, and place on a horizontal shaker at 4°C for 1 h (180 rpm).

[0113] (3) Transfer the mixed system to the chromatography column for filtration. At this time, Protein A Resin is backfilled into the chromatography column, and then 100 mL Bind Buffer is added for further filtration to wash away impurities.

[0114] (4) Add 1 mL of Elution Buffer and collect the filtrate to elute the target protein. Then immediately add 1 / 10 volume (100 μL) of Neutralization buffer to neutralize and mix well to obtain the purified recombinant protein.

[0115] The recombinant protein concentration was determined by SDS-PAGE analysis and BCA method, such as... Figure 4 As shown, SDS-PAGE analysis revealed that the recombinant protein exhibited a single, clear band at approximately 135 kDa, consistent with the theoretical molecular weight (ORF region 130.7 kDa, CD 13102.7 kDa, Fc 25.6 kDa). Furthermore, the protein band was clear and free of impurities, indicating good purity of the recombinant protein.

[0116] Example 3: Specific VNAR Screening 1. Phage library screening: (1) Activation of TG1 cells. TG1 (obtained in Example 1) glycerol bacteria were streaked onto antibiotic-free LB agar plates and incubated overnight at 37°C. The next day, single colonies were picked and inoculated into 5 mL of LB liquid medium and cultured overnight at 37°C with shaking at 220 rpm.

[0117] (2) Coating of immunotubes. ① Experimental group: 100 μg of recombinant protein (obtained in Example 2, 50 μg in the second round) was dissolved in 3 mL PBS; ② Control group: 5 mL of 5% skim milk (skim milk in the library screening was diluted and dissolved in PBS), or 100 μg of recombinant protein with the corresponding label was dissolved in 3 mL PBS as a negative screen. After mixing by pipetting, the mixture was incubated at 37°C for 2 h and then transferred to 4°C for overnight incubation.

[0118] (3) Blocking. The next day, the coating solution in both tubes was aspirated and washed once with 5 mL of PBS solution (discard, shake dry, and aspirate the residual solution). 5 mL of 5% skim milk was added to each tube and the tubes were placed at room temperature for milk blocking.

[0119] (4) Negative screening. After the room temperature sealing begins, take 1 mL of the natural phage library (one round of screening > 1 x 10⁻⁶). 13 PFU / mL, second round screening >1x10 12 The PFU / mL solution was mixed with 1 mL of 5% skim milk, sealed, and then mixed by vertical rotation at room temperature for 1 h. The mixture was then added to the control group immunoassay tubes for negative sieving and incubated at room temperature for 1 h. During this period, the experimental group was kept in a milk-sealed environment for 2 h.

[0120] (5) Preparation of TG1 bacterial culture. Inoculate 20 mL of 2×YT medium with TG1 bacterial culture from the previous day at a ratio of 1%, and culture on a shaker for 2 h until OD reaches 1. 600=0.5. Dispense 10 mL of bacterial solution into two 50 mL sterile Erlenmeyer flasks in advance for later use.

[0121] (6) Positive screening. After negative screening for 1 h, the blocking solution of the experimental group was discarded, and the sample was washed three times with PBS. The phage-milk mixture from the control tube was added for positive screening, and the sample was incubated at room temperature for 1 h. After sealing, the sample was vertically rotated and mixed for 1 h. During this period, the control group was washed three times with PBS, and after being filled with PBS, it was placed at room temperature for 2 h.

[0122] (7) Washing. Discard the liquid in both tubes, add 3 mL of PBST to each tube, seal the membrane and rotate vertically for 5 min to wash (3 washes in the first round of screening and 6 washes in the second round of screening), and then wash twice with PBS.

[0123] (8) Phage elution. Add 1 mL of 0.1 M HCl to each tube, seal the membrane, rotate vertically at room temperature for 8 min to elute the phage, and then immediately add 500 μL of 1 M Tris-HCl (pH=7.4) for neutralization.

[0124] (9) Phage amplification and analysis. The eluted phages were added to 10 mL of TG1 bacterial culture from step (5) and cultured at 37°C with shaking at 220 rpm for 1 h. The experimental and control groups were plated onto counting plates 4, 5, and 6 times, respectively, and incubated overnight at 37°C before counting the colonies. The remaining bacterial culture from the experimental group was centrifuged, plated onto 25×25 cm small iron plates (400 mL solid culture medium), and incubated overnight at 37°C. The next day, the culture was collected to prepare screening phages.

[0125] 2. Phage preparation (1) Colony collection and concentration determination. Add 1-2 mL of sterile 2×YT liquid medium to a 25×25 cm² 2×YT solid medium, scrape off colonies, and collect them into centrifuge tubes. Take 50 μL of bacterial suspension, dilute it 20 times with 950 μL of ultrapure water, and measure the OD using a UV spectrophotometer. 600 .

[0126] (2) Bacterial cell amplification. Based on OD... 600 Take 10 OD of bacterial culture and add 100 mL of 2×YT liquid medium containing ampicillin (initial OD). 600 Incubate at 37℃ with shaking at 200 rpm for 2-3 h until OD reaches 0.1. 600 =0.5.

[0127] (3) Helper phage infection. Add 100 μL of M13K07 helper phage and continue culturing at 37℃ and 220 rpm for 1 h.

[0128] (4) Antibiotic screening. Add kanamycin to a final concentration of 30 μg / mL, lower the temperature to 25℃, and incubate overnight on a shaker at 220 rpm.

[0129] (5) Phage precipitation. The next day, the bacterial solution was centrifuged at 9000×g for 10 min at room temperature to collect the supernatant. 20% volume (about 20 mL) of PEG6000 solution was added, and the mixture was placed on a horizontal shaker at 4℃ and shaken for 1 h (180 rpm). The supernatant was removed by centrifugation at 9000×g for 15 min at 4℃ to obtain a white phage precipitate.

[0130] (6) Phage resuspension and purification. The precipitate was resuspended in 3 mL PBS, centrifuged again (15000×g, 10 min, 4℃) to remove residual cells, and the supernatant was used to determine the OD. 268 (1OD) 268 ≈5×10¹²PFU / mL).

[0131] (7) Phage preservation and use. The prepared first-round phage library can be stored at -20℃ for use in the next round of screening, and the rest should be frozen at -20℃.

[0132] (8) Multiple rounds of screening. Repeat the above phage screening-phage preparation steps 2-4 times until the difference in colony count between the experimental group and the control group is significant (counting ratio > 100), then proceed to the next step.

[0133] 3. Preparation of phage antibodies (1) Add 800 μL of 2×YT medium (containing ampicillin) to each well of a 96-well plate. Using a sterile disposable toothpick, pick a single colony from the last round of screening of the experimental group counting plates and inoculate it into the well. Incubate at 37 °C and 200 rpm with shaking for 4-5 h until OD is reached. 600 ≈0.5.

[0134] (2) Take 110 μL of M13K07 helper phage and add 11 mL of 2×YT medium and mix well. Add 100 μL of the mixture to each well. Continue to incubate at 37℃ and 220 rpm for 1 h with shaking.

[0135] (3) Dissolve 100 μL of kanamycin in 11 mL of 2×YT medium and mix well. Add 100 μL to each well and culture overnight on a shaker at 25°C (220 rpm).

[0136] (4) The next day, centrifuge at 800×g for 20 min at 4℃ for 30 min in a vertical centrifuge. Collect the supernatant containing recombinant phage antibody for subsequent phage ELISA analysis.

[0137] 4.2.3.4 Phage ELISA (1) Protein coating. Prepare two 96-well ELISA plates. Add 100 µL of purified CD13 recombinant protein solution (10 μg protein dissolved in 10 mL PBS) to each well of the experimental group. Add 300 µL of 5% skim milk (dissolved in PBS) or the corresponding labeled negative screening protein to each well of the control group. Incubate at room temperature for 2 h, followed by overnight at 4 °C.

[0138] (2) Milk blocking. The next day, the two ELISA plates were drained and washed once with 1×PBST. 300 µL of 5% skim milk was added to each well of the experimental and control plates and incubated at 37°C for 2 h.

[0139] (3) Primary antibody incubation. Wash both plates once with 1×PBST. Add 100 µL of phage supernatant from a 96-well plate to each well of the experimental and control groups and incubate at room temperature on a horizontal shaker for 2 h.

[0140] (4) Secondary antibody incubation. Wash both plates nine times with 1×PBST. Add 100 µL of Anti-M13 HRP working solution (1:20000 diluted in 1% skim milk) to each well and incubate at room temperature on a horizontal shaker for 1 h.

[0141] (5) TMB color development. Wash both plates nine times with 1×PBST. Add 100 µL of TMB working solution (TMB A:TMBB=1:1) to each well. Incubate at 37℃ for 5-10 min until obvious color development is observed, then add 50 µL of 0.1 M sulfuric acid to terminate the reaction.

[0142] (6) Absorbance measurement and analysis. The OD values ​​of the two plates were measured using a microplate reader. 450 Value. Record the OD of the experimental group. 450 The number of wells was at least five times that of the control group. Plasmids of the corresponding bacterial cultures were extracted from the 96-well plates and sequenced for analysis.

[0143] (7) Phage ELISA verification. After sequencing comparison, sequences with different VNAR amino acids were selected, and the corresponding plasmids were retransformed into TG1 competent cells to prepare phage antibodies and perform phage ELISA verification again.

[0144] Using a natural phage library of the Chinese fan ray, VNARs that specifically recognize the CD13 protein were enriched through two rounds of panning, with CD13 protein (Fc tag) as the experimental group and Fc protein as the control group. The first round of screening conditions were: 100 μg antigen coating and 1 × 10⁻⁶ phages. 13Washed three times with PFU and PBST, the number of single clones in the experimental group was 30 times that of the control group; in the second round of screening, the antigen was reduced to 50 μg and the phage to 1×10⁻⁶. 12 PFU was added and PBST washing was increased to 6 times, and the enrichment fold of monoclonal antibodies in the experimental group was increased to 50-fold. Figure 5 The results showed that, through successive optimization of screening conditions, efficient enrichment of CD13-specific phages was successfully achieved. The binding specificity of the candidate phages was further validated using phage ELISA.

[0145] After the second round of screening, 96 monoclonal antibodies were randomly selected from a counting chamber for phage packaging, and the binding ability of the antibody VNAR displayed on the phage surface to the antigen CD13 was detected by phage ELISA. The OD values ​​of the experimental group and the control group were compared. 450 A ratio >5 was used as the positive criterion, and 18 positive clones were obtained through screening, with a positive rate of 18.75%. The results are as follows: Figure 6 As shown, these positive monoclonal antibodies possess VNAR sequence information targeting CD13, and further analysis of their sequence diversity is needed.

[0146] Extraction of OD in Pahge ELISA assay 450 The 15 positive monoclonal plasmids with the highest ratios were sent to a biological sequencing company for sequencing. The sequencing results were translated and multiple sequence alignment was performed. Based on the differences in amino acid sequences, four different VNAR sequences (A2, E12, C2, C9) were obtained. All four VNAR sequences belong to type IV VNAR (Figure 7).

[0147] Analysis using the ProtParam tool on the ExPASy website revealed that the molecular weights of the four VNARs (A2, C2, C9, and E12) ranged from 11.96 to 12.01 kDa (approximately 1 / 10 the size of traditional antibodies), and their theoretical isoelectric points (pI) were all >7 (Table 13). Furthermore, the average total hydrophilicity coefficients (GRAVY values) of the four VNARs were all negative (range: -0.420 to -0.339), indicating that the four VNAR sequences possess strong hydrophilicity and are less likely to form inclusion bodies during induced expression, thus reducing purification difficulty.

[0148] Table 13 Physicochemical parameters of anti-CD13 VNAR

[0149] Example 4: VNAR eukaryotic and prokaryotic expression 1. VNAR eukaryotic expression The strains used to construct the CD13-specific VNAR recombinant protein expression vector were DH5α competent cells and BL21 competent cells, and the plasmids used were pcDNA3.4-hIgG1Fc and pADL-23C VNAR.

[0150] The construction of the VNAR recombinant protein eukaryotic expression vector requires cloning the VNAR sequence into the pcDNA3.4-hIgG1Fc vector. The VNAR gene sequence was obtained from the comparison of the results of the VNAR-specific screening process in Example 3. The VNAR recombinant protein prokaryotic expression vector is the original plasmid pADL-23CVNAR from the phage library (Example 1).

[0151] Linearization of pcDNA3.4-hIgG1Fc plasmid was performed in the same manner as in Example 2 above.

[0152] VNAR target gene amplification: The VNAR template was obtained from the original plasmid through library screening. The VNAR target fragment was obtained by PCR amplification according to the following reaction system (50 μL). The reaction system is shown in Table 14.

[0153] The PCR reaction procedure is shown in Table 15. After the PCR amplification reaction is completed, immediately place the product on ice to cool. After the temperature drops to room temperature, perform agarose gel electrophoresis, remove the gel block of the current size and position, and purify and recover the product using a gel recovery kit. The product can be stored at -20℃ for subsequent ligation reactions.

[0154] Table 14 VNAR Amplification PCR Reaction System

[0155] Table 15 VNAR Amplification PCR Reaction Procedure

[0156] Ligation and scale-up culture: The VNAR target gene was ligated into the pcDNA3.4-hIgG1Fc vector as described in Example 2, and the ligation system is shown in Table 16. Transformation of the ligation product and identification of the recombinant plasmid were performed as described in Example 2. Subsequent scale-up culture and plasmid extraction were performed as described in Example 1.

[0157] Table 16 Ligation system of pcDNA3.4-hIgG1Fc vector and CD13 fragment

[0158] The eukaryotic expression and purification analysis of VNAR recombinant protein were performed as described in Example 2.

[0159] 2. VNAR prokaryotic expression In addition to eukaryotic expression, the specific VNAR was also purified using BL21(DE3) competent prokaryotic expression. The pure plasmid was transformed into BL21(DE3) competent cells as described in Example 1.

[0160] Recombinant protein expression and purification (His tag): (1) Cell culture and induction of expression: Overnight BL21(DE3) colonies were scraped from solid plates using a pipette tip and inoculated into 5 mL of 2×YT medium (containing ampicillin). The culture was incubated at 37°C with shaking (220 rpm) for 5-6 h until turbidity was observed. The culture was then inoculated at a 1:100 ratio into 200 mL of fresh 2×YT medium and incubated at 37°C with shaking for approximately 2-3 h until OD was reached. 600 =0.5. Add IPTG to the culture medium to a final concentration of 0.5 mM for protein expression induction, adjust the culture temperature to 25℃ and shake (220 rpm) overnight for induction.

[0161] (2) Cell lysis and preparation of crude extract: The next day, the cells were centrifuged at 9000×g for 10 min at 4℃ to collect the cell pellet. The cell pellet was resuspended in 50 mL of Buffer A and mixed well. 500 μL of PMSF (1% of the volume of Buffer A) was added to inhibit protease activity, and the mixture was inverted and mixed well. The cell pellet mixture was fixed on ice and then lysed using an ultrasonic homogenizer. The program was set as follows: frequency 20.0 kHz, power 35%, total time 40 min, ultrasonic on time 3 s, ultrasonic off time 5 s. The lysate after ultrasonic homogenization was centrifuged at 9000×g for 40 min at 4℃, the supernatant was collected and filtered (0.45 μm) to obtain the crude protein extract.

[0162] (3) Ni-NTA affinity chromatography purification: Take 300 μL of Ni-NTA agarose, add 1 mL of PBS, mix well, centrifuge at 8000×g for 1 min at room temperature, discard the supernatant, and bind with the crude extract from the previous step in a vertical spinner at 4℃ for 2 h. Wash the protein purification column with water 4-5 times, add 20 mL of Buffer A, pass the protein mixture through the column for purification, add 100 mL of Buffer A to wash away impurities and perform gradient elution. The specific steps are as follows: 250 mM elution: Seal the bottom of the chromatography column, add 1.5 mL of Buffer A and 1.5 mL of Buffer B mixture and seal the top of the chromatography column. Place the column on a vertical rotator (placed in a 4°C refrigerator) and shake to elute for 30 min. Then filter and collect the protein solution, which is the 250 mM imidazole concentration protein collection solution.

[0163] 500 mM elution: Seal the bottom of the chromatography column, add 1 mL of Buffer B and seal the top of the chromatography column. Place the column on a vertical rotator (placed in a 4°C refrigerator) and shake to elute for 30 min. Then filter and collect the protein to obtain the 500 mM imidazole concentration protein collection solution.

[0164] VNAR antibodies specifically recognizing CD13 protein were expressed and purified using both prokaryotic and eukaryotic expression systems. In the prokaryotic expression system, the pADL-23C original plasmid was transformed into BL21 competent cells, induced by IPTG, and purified using nickel column affinity chromatography to obtain the proteins encoded by all four VNAR antibody sequences (A2, C2, C9, and E13). In the eukaryotic expression system, two VNAR sequences (A2 and C9) with different CDR3 regions were selected and ligated into the pcDNA3.4-hIgG1Fc vector to construct a VNAR-hIgG1Fc fusion protein expression vector. After large-scale extraction of the plasmid, it was transfected into 293F cells, and the proteins encoded by the two VNAR sequences (A2 and C9) were purified by Protein A affinity chromatography. The SDS-PAGE electrophoresis results of the anti-CD13 antibody proteins purified from the prokaryotic and eukaryotic expression systems are shown below. Figure 8 As shown.

[0165] SDS-PAGE electrophoresis analysis revealed that the prokaryotically expressed antibody protein exhibited a single band at 12 kDa, consistent with the theoretical molecular weight, and the band was clear and free of impurities. The eukaryotically expressed antibody protein also showed a single band at 40 kDa, comprising an open reading frame (ORF) of 39.8 kDa, an Fc tag of 25.6 kDa, and C9 protein of 12 kDa. These protein bands were also clear and free of impurities, demonstrating that both prokaryotic and eukaryotic expression systems can efficiently prepare high-purity VNAR antibodies.

[0166] 3. BLI assay for antigen-antibody affinity The affinity of antigen and antibody was assessed using biomembrane interference (BLI) technique. The experimental steps are as follows: (1) Prepare a 10 mg / mL biotin-NHS stock solution dissolved in DMSO and store it at -20℃ for later use.

[0167] (2) The CD13 antigen protein was diluted with PBS to 1 mg / mL, then 10 μL of biotin-NHS stock solution was added, and the mixture was mixed by pipetting and incubated at 4°C for 30-60 min.

[0168] (3) Dialyze the protein with PBS solution overnight, changing the dialysate at least 3 times during the process (the molecular weight cutoff of the dialysis bag should be less than 1 / 3 of the protein molecular weight).

[0169] (4) The concentrations of CD13 antigen and specific antibody were determined again using the BCA method.

[0170] (5) Prepare the reagents required for BLI affinity analysis, including: 0.02% PBST (0.02% Tween-20), CD13 antigen protein (2 μg / mL, diluted with 0.02% PBST), and different concentrations of VNAR (0 nM, 200 nM, 400 nM, 600 nM, all diluted with 0.02% PBST). Add the corresponding solutions to the black 96-well plate in sequence, set the program according to Table 17, and select the SA probe for the reaction. When setting, it is also necessary to change to the SA probe mode. Use Octet Alanysis to analyze the reaction results.

[0171] Table 17 BLI Procedure

[0172] The molecular binding kinetics of CD13 and C9 were detected using Octet K2 software, and the results were analyzed using Octet Data Analysis HT 12 software. First, the CD13 antigen was biotinylated and then immobilized onto an SA biosensor probe. Subsequently, the SA probe was placed in C9 protein solutions of different concentrations, and the binding of CD13 and C9 proteins was analyzed using biolayer interferometry (BLI). Finally, the sensor was transferred to a buffer solution PBST to analyze the dissociation of the C9 antibody protein.

[0173] In the analysis of C9-Fc, three concentration gradients were selected: 200 nM, 400 nM, and 600 nM, with 0 nM as a reference. The system analyzed the changes in the solidified material on the probe and generated real-time binding-dissociation curves. Figure 9 It can be observed that the C9-Fc binding rate is positively correlated with the concentration of the C9 protein solution; after reaching the dissociation phase, its dissociation rate is also positively correlated with the height of the binding signal. The calculated kinetic parameters are as follows: binding constant k on =1.544×10 5 (M*s) -1 dissociation constant K off =2.460×10 -2 (s -1 Affinity constant K D =1.593×10 -7 (M).

[0174] To accurately simulate the molecular docking of the CD13 antigen protein with the VNAR C9 antibody protein, the HADDOCK 2.4 online platform was used for molecular docking. For the CD13 antigen protein sequence, the CD13 structure resolved by X-ray crystallography on the Uniprot platform was used as the template source. The A-chain backbone was extracted using Pymol software, water molecules were removed, and the CD13 protein PDB file was saved as the antibody docking material. For the C9 antibody protein sequence, AlphaFold 3 was used to simulate and predict the C9 protein structure.

[0175] Submit the simulation to the HADDOCK 2.4 online platform for molecular docking simulation, such as... Figure 10 As shown, the antigen CD13 protein appears yellow in the docking diagram and has grooves, while the antibody C9 protein appears pink in the docking diagram and has protrusions, indicating that the antigen and antibody can bind firmly. Comparison of the protein sequence with the molecular docking simulation diagram shows that the C9 sequence mainly binds through the CDR1, HV2, and CDR3 regions.

[0176] In summary, according to embodiments of the present invention, total RNA was isolated from spleen tissue cells of *Syngonium chinense*, reverse transcribed into cDNA, and then amplified using specific primers to obtain VNAR sequences. Subsequently, a natural VNAR phage display library of *Syngonium chinense* was constructed, and the phage library was screened using CD13 protein as an antigen, ultimately successfully identifying four sequences. Prokaryotic and eukaryotic expression of the CD13-specific VNAR sequences were performed, revealing that they are water-soluble and active, allowing for BLI assays to determine antigen-antibody affinity. This significantly reduces antibody development cycle and cost, demonstrating the superior hydrophilicity of *Syngonium chinense*-derived VNAR sequences. The Ka of the CD13 antigen and C9 antibody was measured. D With a value of 159.3 nM, it is at a moderate level of affinity for natural VNAR antibodies; it provides a molecular tool for the development of new CD13 inhibitors and is expected to solve the problem of clinical translation of such inhibitors.

[0177] 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.

[0178] 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 CD13-resistant nanobody of the Chinese fan ray, characterized in that, The amino acid sequence of CDR1 in the complementarity-determining region of the antibody is shown in SEQ ID NO:1, and the amino acid sequence of CDR3 is shown in SEQ ID NO:2 or SEQ ID NO:

3.

2. The anti-CD13 nanobody of the Chinese fan ray as described in claim 1, characterized in that, The frame region of the antibody includes FR1, FR2, FR3a, FR3b and FR4, the amino acid sequence of FR1 is shown in any of SEQ ID NO: 6-9, the amino acid sequence of FR2 is shown in SEQ ID NO: 10, the amino acid sequence of FR3a is shown in SEQ ID NO: 11, the amino acid sequence of FR3b is shown in SEQ ID NO: 12 and the amino acid sequence of FR4 is shown in SEQ ID NO:

13.

3. The anti-CD13 nanobody of the Chinese fan ray as described in claim 1 or 2, characterized in that, The hypervariable ring of the antibody includes HV2 and HV4, the amino acid sequence of HV2 is shown in SEQ ID NO:4, and the amino acid sequence of HV4 is shown in SEQ ID NO:

5.

4. The anti-CD13 nanobody of the Chinese fan ray as described in claim 1, characterized in that, The antibody includes nanobodies with amino acid sequences as shown in any of SEQ ID NO:14-17.

5. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the anti-CD13 nanobody of the Chinese fan ray according to any one of claims 1-4.

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

7. The application of the anti-CD13 Chinese fan ray nanobody as described in claim 1 in the preparation of CD13 inhibitors.

8. A pharmaceutical composition, characterized in that, Including the anti-CD13 nanobody of the Chinese fan ray as described in claim 1.

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