Anti-CD63 protein nano antibody and application thereof

Abstract

The invention discloses a nano antibody for resisting CD63 protein and application of the nano antibody. The antibody comprises complementary determining regions CDR1, CDR2 and CDR3, wherein the amino acid sequences of the CDR1, the CDR2 and the CDR3 are respectively shown as SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4. The nano antibody has high affinity and high specificity to a CD63 protein extracellular domain (CD63-ECD), and the KD of the nano antibody is 48.13 nM. The nano antibody has high affinity, high specificity and excellent stability, is easy to produce, and is beneficial to application in disease diagnosis, drug delivery and scientific research.

Description

Technical Field

[0001] This invention relates to the field of nanobodies, and more particularly to a nanobodies against CD63 protein and their applications. Background Technology

[0002] Antibodies, also known as immunoglobulins, are key effector molecules of the immune system. Synthesized and secreted by B lymphocytes, they can highly specifically recognize and bind to antigenic epitopes, playing an irreplaceable role in disease diagnosis and treatment. Traditional antibodies, such as immunoglobulin G, are tetrameric macromolecules composed of two heavy chains and two light chains linked by disulfide bonds, with a molecular weight as high as approximately 150 kDa. While this complex structure endows them with powerful functions and specificity, it also leads to inherent drawbacks such as complex preparation processes, high costs, and limited stability. More importantly, due to their large molecular size, traditional antibodies exhibit poor tissue penetration, making it difficult to effectively reach target areas such as the interior of solid tumors or cross the blood-brain barrier, which greatly limits their application in in vivo diagnosis and treatment.

[0003] In the 1990s, researchers discovered a unique type of antibody in the blood of camels. These antibodies naturally lack light chains and consist only of heavy chains, hence the name heavy chain antibodies. Their antigen-binding function is handled by a single variable domain, known as a VHH or nanobody. Nanobodies have a molecular weight of only about 15 kDa, about one-tenth the size of traditional antibodies, yet they still possess complete antigen-binding capabilities. Due to their unique single-domain structure, nanobodies exhibit excellent physicochemical properties: including high thermal and chemical stability, maintaining activity under extreme pH and high temperature conditions; excellent solubility and the ability to be efficiently expressed in prokaryotic systems (such as E. coli); and, more importantly, their small molecular weight endows them with excellent tissue penetration capabilities, enabling them to reach targets that are difficult for traditional antibodies to access. Furthermore, the complementarity-determining regions of nanobodies, especially the CDR3 region, are typically longer and have a unique conformation, allowing them to recognize occult epitopes that are inaccessible to traditional antibodies, such as the active site of enzymes or clefts on the surface of viruses. These advantages enable nanobodies to demonstrate enormous application potential in fields such as biosensing, cell imaging, targeted therapy, and diagnostic reagent development, making them an emerging research hotspot in the field of antibody engineering.

[0004] Exosomes are extracellular vesicles with diameters ranging from 30 to 200 nanometers. They are produced by the endosome system within cells and released after fusion with the cell membrane via multivesicular bodies. Enclosed by a phospholipid bilayer, they carry and are enriched with various biologically active molecules, including proteins, nucleic acids (such as mRNA and miRNA), and lipids, playing a crucial role in intercellular communication. In recent years, exosomes have attracted significant attention due to their immense potential in disease diagnosis, prognosis, and treatment. For example, tumor cell-derived exosomes carry specific proteins and nucleic acids associated with their parent cells, making them valuable biomarkers in liquid biopsies. Furthermore, due to their natural biocompatibility and targeting capabilities, exosomes are also considered promising drug delivery carriers.

[0005] However, whether using exosomes as diagnostic biomarkers or therapeutic carriers, efficient and high-purity separation remains a primary and critical technical bottleneck. Currently, exosome separation methods mainly include ultracentrifugation, size exclusion chromatography, polymer precipitation, and immunoaffinity capture. While ultracentrifugation is the gold standard, it is time-consuming, requires expensive equipment, and may damage the integrity of exosomes; polymer precipitation easily co-precipitates impurities; and size exclusion chromatography suffers from co-elution with particles of similar size. Immunoaffinity capture, based on antigen-antibody specific reactions, theoretically possesses the highest specificity, its core being the recognition of marker proteins on the exosome surface.

[0006] Among the numerous surface proteins of exosomes, the four-transmembrane protein family members CD9, CD63, and CD81 are recognized as the most classic exosome markers. CD63, in particular, was the first four-transmembrane protein to be characterized, exhibiting high specificity and expression on the exosome membrane, making it an ideal target for identifying and isolating exosomes. Currently, immunocapture targeting CD63 mainly relies on traditional monoclonal antibodies. Despite their effectiveness, the inherent large molecular weight, relatively poor stability, and high production cost of traditional antibodies limit their application efficacy in conditions requiring rapid, low-cost, or demanding settings (such as industrial exosome purification and rapid bedside detection).

[0007] Therefore, developing a novel binding molecule that can specifically target CD63 while possessing advantages such as high affinity, high stability, and low production cost is of vital importance for promoting basic research and clinical translation related to exosomes. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of traditional anti-CD63 antibodies in terms of stability, production cost, and tissue penetration, and to provide an anti-CD63 nanobody with high affinity, high specificity, excellent stability, and easy production, as well as its complete set of technical solutions.

[0009] To achieve the above objectives, the present invention provides a nanobody characterized in that it comprises complementarity-determining regions CDR1, CDR2 and CDR3, wherein the amino acid sequences of CDR1, CDR2 and CDR3 are as shown in SEQ ID NO: 3, 4 and 5, respectively.

[0010] Furthermore, the nanobody has an amino acid sequence as shown in SEQ ID NO: 1.

[0011] The present invention also provides a nucleotide, characterized in that it encodes the nanobody.

[0012] Furthermore, the nucleotide sequence is shown in SEQ ID NO: 2.

[0013] The present invention also provides an expression vector, characterized in that it comprises the aforementioned polynucleotide.

[0014] The present invention also provides a host cell, characterized in that it contains the nucleotides or the expression vector.

[0015] The present invention also provides a method for preparing the nanobody, characterized in that it includes: culturing the host cells and isolating and purifying the nanobody from the culture.

[0016] The present invention also protects the use of the nanobody in binding to the CD63 protein.

[0017] The present invention also provides a kit for detecting or binding CD63 protein, characterized in that it comprises the nanobody.

[0018] This invention also protects the use of the nanobody in the preparation of products for the separation, detection or targeting of exosomes.

[0019] Furthermore, the product is an immunoaffinity chromatography medium, an ELISA detection reagent, an immunofluorescence detection reagent, or an in vitro diagnostic device.

[0020] The present invention also provides a product for separating, detecting or targeting exosomes, characterized in that it comprises the nanobody.

[0021] Furthermore, the product is an immunoaffinity chromatography medium, an ELISA detection reagent, an immunofluorescence detection reagent, or an in vitro diagnostic device.

[0022] The high-performance nanobody targeting CD63 described in this invention can replace or supplement traditional antibodies, achieving a technological breakthrough in the fields of exosome separation, detection, and targeted applications.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] 1. High affinity and specificity: The nanobody 2E provided by this invention has an equilibrium dissociation constant (KD) for the extracellular domain of CD63 protein at the nanomolar level (48.13 nM), and has been verified by cellular level and gene knockout model, showing high specificity.

[0025] 2. Small molecular weight and strong penetration: Its small molecular weight (about 15 kDa) makes it more advantageous in terms of tissue penetration and antigen epitope recognition, especially suitable for deep tissue imaging or complex sample detection.

[0026] 3. Good stability: Nanobodies have better thermal and chemical stability than traditional antibodies, making them easier to store and transport, and able to adapt to more demanding application environments.

[0027] 4. Easy to produce and modify: It can be expressed in large quantities and at low cost in prokaryotic systems, and is easy to genetically engineer (such as constructing multivalent, multispecific antibodies or fusion proteins), with flexible and diverse applications. Attached Figure Description

[0028] Figure 1 This is a graph showing the enrichment results of the anti-CD63 nanoantibody screening.

[0029] Figure 2 This is an ELISA result of the binding activity of nanobody 2E to CD63-ECD.

[0030] Figure 3 This is an SDS-PAGE image of the purified nanobody 2E obtained in this embodiment of the invention, stained with Coomassie brilliant blue.

[0031] Figure 4 This is a biofilm layer interference (BLI) sensor image and kinetic parameters of nanobody 2E bound to CD63-ECD.

[0032] Figure 5 This is an immunofluorescence colocalization analysis showing the specific binding of nanobody 2E to the CD63 protein on the surface of 293T cell membranes.

[0033] Figure 6 This is a diagram showing the results of verifying the specific binding of nanobody 2E and CD63 in 293T cells in Example 4. Detailed Implementation

[0034] The embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0035] In the following embodiments: 2×YT liquid medium: 200 ml; 2×YT liquid medium: 3.2 g peptone; 2 g yeast extract; 1 g NaCl; 2×YT agar plate: 200 ml 2×YT agar plate: 3.2g peptone; 2g yeast extract; 1g NaCl; 3g agar powder.

[0036] Example 1: Screening and Identification of Anti-CD63 Nanobodies Biological panning of camel-derived VHH phage display library using purified human CD63-ECD protein (SEQ ID NO:6) as a target.

[0037] Nucleic acid sequence of human CD63-ECD protein (303 bp): GCTGGCTATGTGTTTAGAGATAAGGTGATGTCAGAGTTTAATAACAACTTCCGGCAGCAGATGGAGAATTACCCGAAAAACAACCACACTGCTTCGATCCTGGACAGGATGCAGGCAGATTTTAAGTGCTGTGGGGCTGCTAACTACACAGA TTGGGAGAAAATCCCTTCCATGTCGAAGAACCGAGTCCCCGACTCCTGCTGCATTAATGTTACTGTGGGCTGTGGGATTAATTTCAACGAGAAGGCGATCCATAAGGAGGGCTGTGTGGAGAAGATTGGGGGCTGGCTGAGGAAAAATGTG. SEQ ID NO: 6.

[0038] 1. VHH Selection: (1) Take two immunoassay tubes and set up an experimental group (tube A) and a control group (tube B); Experimental group (tube A): 500 μg of purified human CD63-ECD protein was diluted to 5 mL with PBS and added to tube A of experimental group. The mixture was incubated overnight at 4°C. The liquid in tube A was then discarded, and 5 mL of blocking buffer was added. The mixture was blocked at 37°C for 2 h. The blocking buffer in tube A was then discarded. Control group (tube B): 5% skim milk powder (prepared with PBS) was added to tube B of the control group and incubated overnight at 4°C; then washed once with PBST; the pretreated phage solution was added to tube B and incubated for 1 h; The pretreated phage solution was prepared by mixing 100 μL of camel-derived VHH phage library with 2 mL of 5% skim milk powder and incubating at room temperature for 1 h. The camel-derived VHH phage library was prepared by extracting RNA from the blood of young, healthy camels. Following the instructions of the reverse transcription kit (ABclonal), the RNA was reverse transcribed into complementary DNA (cDNA). Two rounds of amplification were then performed on this cDNA to obtain a specific antibody fragment (VHH gene). The primers used for amplification were: Round F was GTCCTGGCTGCTCTTCTACAAGG (SEQ ID NO:10), and Round R was GGTACGTGCTGTTGAACTGTTCC (SEQ ID NO:11); Round F-1 was TATAATGCCCAGCCGGCCATGGCAGATGTGCAGCTGCAGGAGTCTGGAGGAGG (SEQ ID NO:12), Round F-2 was TATAATGCCCAGCCGGCCATGGCAGATGTGCAGCTGCAGGAGTCTGGGGGAGG (SEQ ID NO:13), and Round R was AATTAGCCTCCCGGGCCTGAGGAGACGGTGACCTGGGT (SEQ ID NO:14). Restriction endonucleases were used. Bgl I. The phage display vector pADL-23C and its amplification products were digested with enzymes and ligated to form a complete circular plasmid, which was then transformed into TG1 competent cells. The bacterial culture was cultured to OD100. 600 =0.5-0.55, then add helper phage M13KO7 (≥1×10). 13 PFU / mL) was used to assist phage infection. After culturing, the bacterial cells were removed to obtain the supernatant. The supernatant precipitate was resuspended and concentrated to obtain the purified phage library.

[0039] (2) Transfer all the phage suspension in tube B to tube A. Incubate at 25°C for 60 min to allow the phage to fully bind to the target. At the same time, add 5 mL of phosphate-buffered saline (PBS, pH 7.4) to the empty tube B for equilibration. (3) Remove the supernatant from tubes A and B respectively, add PBST washing buffer containing 0.1% Tween-20 to each tube, seal the tube opening, and place them on a reverse mixer to wash at 30 rpm for 5 min. Repeat this washing process 3 times. (4) Add 1 mL of 0.1 mol / L hydrochloric acid solution (HCl) to tubes A and B respectively, and treat them at room temperature for 8 min to dissociate the bound phages. This step requires strict control of the time to ensure the integrity of the phages. (5) Then immediately add 500 μL of 1 M Tris-HCl (pH 7.4) to neutralize; (6) Pick E. coli TG1 single clones (purchased directly) in advance and put them into a 100 mL Erlenmeyer flask containing 20 mL of 2 × YT liquid medium. Incubate at 37 °C on a horizontal shaker at 250 rpm until the OD600 is about 0.5. Take 10 mL of bacterial culture and dispense it into two 50 mL Erlenmeyer flasks. Add the two sets of eluted phage libraries into them respectively. Incubate in a constant temperature horizontal shaker at 37 °C and 200 rpm for 1 h.

[0040] (7) Gradient dilution (10 -4 and 10 -5 Afterwards, the bacteria were plated and cultured; the remaining experimental bacteria were centrifuged at room temperature, some of the supernatant was removed, and the bacteria were resuspended and plated onto 2 × YT solid iron plates containing 100 µg / mL ampicillin for the collection of selected phages. (8) Collect the colonies formed on the surface of the solid culture medium on the iron plate, suspend them evenly, and then transfer them to 2×YT liquid medium containing 100 μg / mL ampicillin. Adjust the initial optical density (OD600) to 0.08-0.12. Place in a 37℃ constant temperature shaking incubator and incubate at 250 rpm until the logarithmic growth phase (OD600=0.5). (9) After a single wash with PBST (containing 0.05% Tween-20), tube A of experimental group was incubated with M13KO7 helper phage at 37°C with shaking for 60 min. Then, kanamycin was added to a final concentration of 50 μg / mL, the incubation temperature was adjusted to 25°C, and the tubes were incubated at 220 rpm for 14-16 hours. (10) Centrifuge the culture at 7,800 rpm for 15 min at 4°C and collect the supernatant. Add PEG-NaCl precipitant equivalent to 20% of the supernatant volume and gently shake on ice for 60 min to promote phage particle aggregation; (11) Centrifuge at 4°C and 12,000 rpm for 15 min to collect the precipitate, and gently resuspend the particles in 1 mL of pre-cooled PBS buffer (pH 7.4). Centrifuge again under the same conditions for 5 min to remove residual impurities. The collected supernatant is the purified phage display library. (12) Using a 10-fold serial dilution method, 10 μL of each dilution was mixed with 990 μL of logarithmic-phase TG1 Escherichia coli (OD600=0.5), incubated at 37℃ for 60 min, and then plated on 2×YT agar plates. Plaque counting was performed after incubation at 37℃ for 16 hours. The purified phage particles were then subjected to two more rounds of increasingly stringent screening experiments. The specific screening parameters are detailed in Table 1.

[0041] Table 1. Parameter configuration for each round of phage display screening.

[0042] 2. Phage ELISA for panning specific VHH monoclonal antibodies: Day 1 Operation Procedure: (1) Inject 800 μL of 2×YT liquid culture medium containing 100 μg / mL ampicillin into each well of a 96-well deep-well culture plate. Use a sterile toothpick to pick up the monoclonal colonies on the experimental group counting plate and inoculate them into the corresponding wells. Cover with a sterile breathable sealing film and place in a 37°C constant temperature shaker and culture at 250 rpm. (2) Mix M13KO7 helper phage (MOI=20) with 2×YT medium at a volume ratio of 1:100 to obtain a mixture, and add 100 μL of this mixture to each well. Continue to incubate at 37℃ with shaking for 60 minutes to promote phage adsorption; (3) Add 100 μL of 2×YT medium containing 50 μg / mL kanamycin sulfate to each well. Incubate at 250 rpm for 16-18 hours in a constant temperature shaker at 250°C to complete phage packaging; (4) For the experimental plates, CD63-ECD protein was diluted to 500 ng / well (final concentration 5 μg / mL) with PBS, and 100 μL was coated in each well. The plates were then incubated at 4°C for 16 hours. For the control plates, 300 μL of PBS blocking buffer containing 5% skim milk was added to each well, and the plates were blocked at room temperature for 16 hours.

[0043] Next-day testing process: (1) Discard the coating solution, and rinse both the experimental plate and the control plate once with PBST. Add 300 μL of blocking solution containing 5% skim milk to each well and incubate at 37°C for 2 hours; (2) Centrifuge the 96-well deep-well culture plate that has been cultured overnight at 4℃ and 4,500 rpm for 5 min, collect the supernatant and store it temporarily at 4℃ for later use; (3) After washing the ELISA plate 3 times with PBST, add 100 μL of the supernatant after centrifugation to each well, incubate at room temperature for 120 min, wash 9 times with 1×PBST, add 100 μL of HRP-labeled anti-M13 antibody (diluted with blocking buffer) diluted 1:20,000 to each well, and incubate at room temperature for 60 min. (4) Add 100 μL of TMB substrate solution to each well. After 5 minutes of colorimetric reaction, add 50 μL of 0.1M sulfuric acid to stop the reaction. Immediately measure the absorbance at 450 nm using a microplate reader. (5) Select positive wells with OD450 values ​​significantly higher than 3 times that of the control, and extract plasmids from the corresponding bacterial culture after expansion culture and send them to the biotechnology company for sequencing and data analysis.

[0044] Results Explanation: This study used phage display technology to screen a camel-derived nanobody library, targeting CD63 protein. High-affinity, specific nanobodies were successfully enriched through three rounds of increasingly stringent panning. During the three rounds of panning, the antigen coating concentration was successively decreased from 100 μg / mL to 50 μg / mL and then to 25 μg / mL, while the number of washes increased from 3 to 6, gradually increasing the screening pressure. The results showed that the enrichment ratio increased significantly with each screening round (…). Figure 1 This indicates that phages that specifically bind to CD63 were effectively enriched.

[0045] Validated by phage ELISA, 55 out of 96 randomly selected monoclonal antibodies showed a positive reaction, with a positive rate of 57.89%. Figure 2 (A). Sequencing of positive clones yielded six unique VHH sequences, named 1A, 1E, 2E, 4A, 5D, and 9C (see A). Figure 2Based on the CDR3 region sequence characteristics, these nanobodies are divided into three families: Family I (1A, 1E, 5D) has a CDR3 region rich in aromatic amino acids (W / Y / F) and a similar charge distribution pattern; Family II (2E, 4A) has a CDR3 region dominated by hydrophobic residues (V / M / I) and proline (P), which may form a rigid structure; Family III (9C) has a unique CDR3 region composition, which may represent a novel binding mode.

[0046] Sequence analysis revealed that families I and II likely originated from the same germline gene, with their functional differences primarily due to sequence differentiation in the CDR3 region. The FR1 and FR2 initiation regions of all sequences were highly conserved, and the FR3b region was completely identical, suggesting that this region plays a crucial role in maintaining structural stability. Furthermore, all VHHs exhibited typical Camelidae nanobody characteristics in the FR2 region, namely, hydrophobic residues (such as Gly44 and Leu45) were replaced by hydrophilic residues (such as Phe / Gln), which contributes to enhanced solubility. Notably, nanobody 2E contains multiple aromatic residues (Y / W) in its CDR3 region, which are presumably enhanced with antigen binding through hydrophobic interactions, suggesting potentially high affinity and providing an important candidate sequence for subsequent functional studies. The complete amino acid sequence of nanobody 2E is shown in SEQ ID NO: 1.

[0047] MAEVQLQASGGGFVQPGGSLRLSCAASGRTYKRTGMGWFRQAPGKEREFVSAISEGDSTAPYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCADYPQSRAVMRHWHRWWQGYWGQGTQVTVSS. SEQ ID NO:1.

[0048] The nucleotide sequence corresponding to nanobody 2E is shown in SEQ ID NO:2: ATGGCAGAAGTTCAGCTGCAGGCAAGCGGTGGTGGTTTTGTTCAGCCTGGTGGTAGCCTGCGTCTGAGCTGTGCAGCCAGCGGTCGTACCTACAAACGTACCGGTATGGGCTGGTTTCGCCAGGCACCGGGTAAAGAACGTGAATTTGTTAGCGCAATCAGCGAAGGTGATTCTACTGCACCATATTATGCCGAT AGCGTGAAAGGTCGCTTTACCATTAGCCGTGATAATAGCAAAAATACCGTTTACCTGCAGATGAATAGTCTGCGTGCAGAAGATACCGCAACCTATTATTGTGCAGATTACCCACAGTCTCGTGCAGTTATGCGTCATTGGCATCGTTGGTGGCAGGGTTATTGGGGTCAGGGCACCCAGGTTACCGTTAGCAGC. SEQ ID NO: 2.

[0049] CDR1: SGRTYKRTGM. SEQ ID NO: 3; CDR2: SEGDSTAP. SEQ ID NO: 4; CDR3: DYPQSRAVMRHWHRWWQG. SEQ ID NO: 5.

[0050] Example 2: Prokaryotic Expression and Purification of Nanobodies 2.1. The bacterial culture of the positive clones (55 positive clones) obtained in Example 1 was transferred at a volume ratio of 1:100 to a test tube containing 5 mL of 2 × YT liquid medium, and ampicillin was added to a final concentration of 100 µg / mL. The culture was incubated at 37 °C in a horizontal shaker for 12–16 h. Plasmid 2E carrying the nanobody gene was extracted using a plasmid miniprep kit (Tiangen Biotech), and the plasmid concentration was measured using Nanodrop.

[0051] 2.2. Plasmid Transformation (1) Take out E. coli WK6 competent cells from the -80 °C ultra-low temperature freezer, place them on ice for about 6 min until the bacterial block melts, add 100 µg of the previously extracted plasmid with nanobody gene, gently tap the bottom of the centrifuge tube to mix, and let stand on ice for 20 min.

[0052] (2) Transfer the centrifuge tubes to a 42 °C water bath for 90 s heat shock, and then quickly put them back into ice and let them stand for 5 min.

[0053] (3) Add 900 µL of LB liquid culture medium to the centrifuge tube and revive it at 220 rpm for 45 min in a horizontal shaker at 37 °C.

[0054] (4) Centrifuge at 7,000 rpm for 2 min to collect the bacteria, and collect about 100 µL of supernatant. Gently pipette the bacterial block to resuspend it and spread it onto an LB solid medium plate containing ampicillin resistance (100 µg / mL). Incubate the plate upside down at 37 °C overnight.

[0055] 2.3. Induced Expression of Nanobodies TB liquid culture medium: glycerol 20 mL / L, yeast extract 24 g / L, tryptone 12 g / L (1) Pick a single colony from an LB plate containing 100 μg / mL ampicillin and inoculate it into 5 mL of TB liquid medium. Incubate at 37°C and 220 rpm for 12-16 h with shaking. (2) Take 2 mL of the above-mentioned seed culture in the logarithmic growth phase (OD600≈0.8) and transfer it to 200 mL of freshly prepared TB liquid culture medium. Incubate in a constant temperature shaker at 37℃ with shaking at 250 rpm. Dynamically monitor the OD600 value of the culture medium using a spectrophotometer. When it reaches 0.8, enter the induction phase. (3) Add IPTG to a final concentration of 1 mM, and culture at 28℃ and 220 rpm for 16 h to induce recombinant protein expression; (4) Transfer the induced bacterial culture to a pre-cooled centrifuge tube and centrifuge at 7,800 rpm for 20 min at 4°C. Discard the supernatant containing metabolic byproducts. (5) Resuspend the bacterial cells in 5 mL of TES buffer (20 mM Tris-HCl, 1 mM EDTA, 20% sucrose, pH 8.0) and shake on ice for 60 min; (6) Add 5 mL of 0.5 mM MgSO4 solution and continue shaking in an ice bath for 30 min; (7) Centrifuge at 12,000 rpm for 15 min at 4℃ and collect the supernatant. Filter the supernatant through a 0.45 μm filter membrane to obtain crude protein extract, and store at -80℃ for later use.

[0056] 2.4. SDS-polyacrylamide gel electrophoresis Sample preparation: Collect 20 μL of eluent, add protein 4× SDS loading buffer, and boil at 100℃ for 10 min. Centrifuge at 10000 rpm / min for 1 min, then place on ice. Prepare a separating gel with a concentration of 12%; load the sample, electrophoresis at a constant voltage of 80 V for 30 min, then increase the voltage to 120 V and continue electrophoresis for 1 h. Remove the protein gel, add an appropriate amount of Coomassie brilliant blue staining solution, and stain at room temperature on a shaker for 1 h. Destain overnight with destaining solution, and photograph and observe the target protein bands the next day.

[0057] 2.5. BCA Protein Quantification (1) Take out the bovine serum albumin (BSA) standard with a concentration of 2 mg / mL from the storage conditions of -20℃, equilibrate at room temperature until completely thawed, and gently vortex to mix for later use; (2) Prepare five clearly labeled 1.5 mL centrifuge tubes, labeled with concentrations of 2.0, 1.0, 0.5, 0.25, and 0.125 mg / mL respectively. Prepare standard solutions of each concentration gradient by double dilution using phosphate-buffered saline (PBS) at pH 7.4 or the corresponding experimental buffer system. (3) Mix solution A (alkaline copper solution) and solution B (BCA chelating agent) in the BCA test kit at a ratio of 50:1 (v / v), mix thoroughly, and store away from light. Prepare and use immediately. (4) Add 200 μL of BCA working solution to each well of the 96-well plate; (5) Add the standard solution and the sample to be tested in sequence; (6) Incubate the 96-well plate at room temperature in the dark for 30 min. Measure the absorbance (OD value) of each well using a microplate reader at a wavelength of 562 nm. Plot a standard curve (linear regression fitting) with the standard concentration as the x-axis and the OD562 value as the y-axis. Calculate the protein concentration based on the sample OD values.

[0058] 2.6. Dialysis of protein solutions (1) Material preparation: Select a regenerated cellulose dialysis membrane with a suitable molecular weight cutoff and cut it to a working length of 15 cm. Completely immerse the dialysis membrane in boiling deionized water and sterilize it at 100°C for 10 min; (2) Rinse the inner and outer surfaces of the dialysis membrane alternately five times with Milli-Q ultrapure water, each rinse lasting 30 seconds, to ensure removal of the glycerol coating and sterilization residue. Secure one end of the membrane tubing with a dialysis clamp to form a sealed chamber. Invert the device to check for airtightness and confirm there is no risk of leakage. (3) Use a flat-mouth pipette to slowly inject the protein solution to be dialyzed along the tube wall to avoid the formation of air bubbles. After the injection is complete, expel the air at the top and clamp the open end again. Pre-cool 1 L of PBS buffer and vertically immerse the sealed dialysis apparatus in the dialysate; (4) Set the magnetic stirrer speed to 80~90 rpm and maintain the solution temperature at 4℃; (5) Replace the pre-cooled fresh dialysate every 120 min, and perform a total of 3-4 complete replacements; (6) Under aseptic conditions, open the dialysis clamp and use a blunt-tipped pipette to transfer the sample to a centrifuge tube. Centrifuge at 12,000 rpm for 120 s at 4°C to remove trace amounts of precipitate, and store at 80°C for long-term storage.

[0059] Results Explanation: In the above study, we successfully screened nanobodies with anti-CD63 activity. To obtain these predicted nanobodily proteins, we transformed the gene fragment encoding the target nanobodies into *E. coli* WK6 competent cells, extracted and purified the protein. The purified protein was analyzed by SDS-PAGE electrophoresis and Coomassie brilliant blue staining. The results showed a clear band at approximately 15 kDa, indicating successful expression of the target protein. Figure 3 The obtained nanobody protein samples will be further used for functional verification experiments on antigen-antibody binding.

[0060] Example 3: Determination of binding affinity and kinetics of nanobodies 3.1 Enzyme-linked immunosorbent assay (ELISA) was used to verify the affinity of 2E (the same applies to 5D and 1A methods), as follows: (1) Antigen dilution and coating: Dissolve the antigen in phosphate buffer (10 mM PBS, pH 7.4) and adjust the final concentration to 1.5 μg / mL. Accurately add 100 μL of diluted antigen to each well of a 96-well high-binding enzyme-linked immunosorbent assay (ELISA) plate, cover the plate with the membrane, and incubate at 4°C for 12-16 hours. (2) After pouring out the coating solution, add 300 μL of 1×PBST to each well and wash once. Add 300 μL of 5% skim milk powder (prepared with PBS) blocking solution to each well and block at 37℃ for 2 h. (3) Discard the blocking buffer, wash once with 1×PBST, add the antibody to be tested (diluted serially with 2.5% skim milk powder) to each well of the experimental group, and do not add it to the blank group. Add 100 μL to each well of both the experimental and control groups, and incubate at room temperature for 2 h; (4) Wash 9 times with 1×PBST, dilute Anti-His-HRP antibody with 2.5% skim milk powder at a ratio of 1:10,000, mix well and add 100 μL to each well, and incubate at room temperature with shaking for 1 h; (5) Wash 9 times with 1×PBST, add 100 μL of TMB colorimetric solution to each well, and develop the color at room temperature in the dark for 5~10 min. When there is a clear color change, immediately add 50 μL of 0.1 M H2SO4 to each well to stop the reaction. (6) The absorbance value is measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0061] 3.2 The determination of 2E (and 5D and 1A methods) binding capacity based on biolayer interferometry (BLI) is as follows: (1) Dilute the CD63-ECD antigen to a final concentration of 1.0 mg / mL with 10 mM PBS (pH 7.5). Dissolve NHS-LC-Biotin in anhydrous DMSO to prepare a 10 mM stock solution; (2) Add 4.54 μL of biotinylated reagent stock solution to 1 mL of antigen solution (molar ratio 10:1), and place in a 25℃ dark environment with gentle shaking for 25 min. During this period, use a vortex mixer (1000 rpm, 10 s) every 10 min to ensure uniform mixing; (3) Use a dialysis membrane with a molecular weight cutoff of 3.5 kDa to dialyze PBS buffer at 4°C for 12 hours. The volume of dialysate is 500 times the volume of the sample. Replace the dialysate with fresh dialysate every 4 hours. (4) Dilute the antibody with PBST buffer containing 0.02% Tween-20 to create a concentration gradient, and set up a blank control (buffer only); (5) Using the Octet RED96e Molecular Interaction Analyzer and its accompanying Discovery Software, set the standard detection program as shown in Table 2: (6) Add 200 μL of the corresponding solution to each well of the black 96-well plate and run the program; (7) Use Octet Analysis 12.2 software to process the raw data and use a 1:1 combined model fitting to calculate the binding rate constant (ka), dissociation rate constant (kd) and equilibrium dissociation constant (KD).

[0062] Table 2. Procedure of the Biomolecular Interaction Analyzer

[0063] Results Explanation: Systematic screening of a phage display library specific to the CD63 extracellular domain (CD63-ECD) successfully yielded and expressed three candidate nanobodies (5D, 2E, and 1A). To systematically evaluate the specific binding ability of these nanobodies to CD63-ECD, an enzyme-linked immunosorbent assay (ELISA) was performed: recombinant CD63-ECD protein was immobilized in 96-well plates and incubated with purified nanobodies 5D, 2E, and 1A, respectively. Skim milk was used as a control group. The HRP-TMB colorimetric system was used for detection, and the absorbance (OD450) of each well was measured at 450 nm using a microplate reader. Experimental data (Figure 4a) showed that, compared with the negative control group, nanobodies 5D and 2E exhibited significant antigen-binding activity, while no obvious binding signal was detected for 1A. Notably, the binding signal intensity of the 2E nanobody was significantly superior to that of the 5D nanobody, with an OD450 value approximately 3.1 times higher than that of 5D, indicating that 2E has a higher affinity for CD63-ECD. This result confirms that the 2E nanobody prepared via a eukaryotic expression system possesses the best antigen-binding performance. Therefore, we selected the 2E recombinant protein with the best binding performance to conduct a systematic in vitro binding characteristic analysis.

[0064] The experiment employed a standardized enzyme-linked immunosorbent assay (ELISA). CD63-ECD was coated and immobilized on a solid-phase support, and 2E antibody was incubated with 13 consecutive dilution gradients (2.5-500 nM). Skim milk was used as a negative control. In this experiment, the signal intensity represents the amount of 2E antibody bound to CD63-ECD. Experimental data (Figure 4b) showed that as the concentration of 2E antibody increased, OD450 increased from 0.0715 to 0.5698, and the detection signal intensity exhibited a typical dose-dependent increasing trend, fully confirming the specific binding characteristics of 2E to CD63-ECD. Further nonlinear regression analysis of the experimental data yielded a half-maximal effective concentration (EC50) of 2E antibody binding to CD63-ECD of 0.5046 nM. This value is significantly lower than the EC50 range (1-10 nM) of conventional nanobodies, indicating that 2E has an extremely high binding affinity for CD63-ECD (Figure 4c).

[0065] After initially verifying the binding activity of the 2E recombinant nanobody with CD63-ECD, this study systematically and quantitatively analyzed the molecular interaction kinetics of the two molecules using highly sensitive bio-layer interferometry (BLI). The Octet K2 molecular interaction analysis system (ForteBio) was used to accurately determine the binding and dissociation processes between protein molecules by real-time monitoring of the wavelength shift of the interference light caused by changes in the thickness of the biofilm on the surface of the amino-coupled sensor. In the kinetic analysis experiment, four concentration gradients (50 nM, 250 nM, 550 nM, and 750 nM) of the 2E nanobody solution were set up, with PBS buffer as a blank control. A 1:1 binding model was used to perform nonlinear regression fitting analysis on the obtained binding-dissociation curves (Figure 4, d). Experimental data show that during the binding phase, the binding signal response value (RU) exhibits a good dose-dependent relationship with antibody concentration; during the dissociation phase, high-concentration samples show a faster dissociation rate, forming a typical "rapid binding-moderate dissociation" kinetic characteristic, which is beneficial for achieving an efficient target capture and release balance. The kinetic parameters obtained through global fitting analysis (Figure 4e) show that the binding rate constant (Ka) is 5.805 × 10⁻⁶. 4 M - ¹s - ¹, The dissociation rate constant (Kd) is 2.794 × 10⁻⁶. - ³ s - ¹, Based on this, the equilibrium dissociation constant (KD) calculated is 4.813 × 10¹⁰. -8 M. These data indicate that the 2E nanobody has a high affinity for CD63-ECD.

[0066] Based on the above experimental results, these quantitative analyses are highly consistent with the previous screening data, confirming not only the high affinity properties of the 2E nanobody but also providing important dosage references for subsequent functional studies. Therefore, this study identifies 2E as the most promising candidate antibody molecule for development. Future research will focus on conducting systematic studies on this antibody to provide solid experimental evidence for its application in diagnostics or therapy.

[0067] Example 4: Verification of the binding specificity of nanobody 2E 4.1. Construction of the CV296-CD63 fusion protein vector Using the primers in Table 3, CD63 fragments with homologous arms at both ends were obtained by PCR amplification. The recovered CD63 gene fragment was then mixed with the enzyme-digested and recovered CV296 vector (commercially available, such as from Heyuan Biotechnology (Shanghai) Co., Ltd.) in a 5×TEDA ligase (1 mL 5×TEDA buffer, 1 μL T5 Exonuclease) ligation buffer system and incubated at 30℃ for 40 min to complete the ligation of the CD63 fragment and the CV296 vector. The steps are as follows: 4.1.1. CV296 plasmid digestion use Nhe I and Xho I. Prepare 50 μL reaction systems with restriction endonucleases according to the optimized ratios shown in Table 4. Incubate the reaction systems with the corresponding restriction endonucleases in a 37°C water bath for 120 min, gently mixing the reaction systems every 30 minutes.

[0068] Table 4 Enzyme digestion reaction system

[0069] 4.1.2. CD63 Full-Length Gene Amplification The full-length CD63 gene fragment (lysosome-associated membrane protein 3, LAMP-3) was amplified according to the following PCR system. The PCR system is shown in Table 5, and the PCR reaction procedure is shown in Table 6.

[0070] Table 5 PCR Reaction System

[0071] The primer sequences are as follows: Table 3 Primer sequence listing

[0072] Template: Human CD63 full-length gene sequence (714 bp): CATCACCTCGTAGCCACTTCTGATACTCTTCACGAGGCAGCAGGCAAAGACAATTCCCAAAACCTCGACAAAAGCAATTCCAAGGGCTGCTGCAGCTACCACCAGCACATTTTTCCTCAGCCAGCCCCCAATCTTCTCCACACAGCCCTCCTTATGGATCGCCTTCTCGTTGAAATTAATCCCACAGCCCACAGTAACATTAATGCAGCAGGAGTCGGGGACTCGGTTCTTCGACATGGAAGGGATTTTCTCCCAATCTGTGTAGTTAGCAGCCCCACAGCACTTAAAATCTGCCTGCATCCTGTCCAGGATCGAAGCAGTGTGGTTGTTTTTCGGGTAATTCTCCATCTGCTGCCGGAAGTTGTTATTAAACTCTGACATCACCTTATCTCTAAACACATAGCCAGC AATGGCTGCGGCCACCTCCACCAACATGATAAGAGACAGAAAGATGGCAAACGTGATCATAAGACAATAGTTCTCCTTGCAGGCCCCGCAGCAGCCCACAAAAGCCACCAGGAAGAGGAAGACACCCACTGCGATGATGACCACTGGCAACAGAGAGCCAGGGGTAGCCCCCTGGATTATGGTCTGACTCAGGACAAGCTGTGCCCCGACACCCACGGCAATCAGTCCCACTGCACAGGCGCAAAAGGCCAGCAGGAGGACGTAGAGCAAGAACTTCACACATTTCATTCCTCCTTCCACCGCCAT. SEQ ID NO:9。

[0073] Table 6 PCR reaction program table

[0074] 4.1.3. Agarose gel electrophoresis of DNA Weigh 1.4 g of agarose and add 141 mL of TAE buffer solution (purchased from Sangon Biotech). Microwave on high for 2.5 min until the solution is clear. Add nucleic acid dye and pour into an agarose gel for electrophoresis. Mix the PCR amplification product with loading buffer and load the sample. Electrophoresis is performed at a constant voltage of 120 V for approximately 30 min, stopping when the bromophenol blue line reaches the lower part of the gel. Irradiate the gel with UV light, and then carefully cut the target band from the agarose gel using a scalpel blade for subsequent experiments. The gel can be temporarily stored at -20°C in the dark.

[0075] 4.1.4. DNA Recovery from Agarose Gel (1) Take the gel out of the refrigerator, weigh it, add the solution PC and put it directly into a water bath at 50°C until the colloid is completely dissolved.

[0076] (2) Equilibrate the adsorption column. Assemble the adsorption column and the collection tube, then add 500 μL of BL equilibration solution. Centrifuge at 12,000 rpm for 1 min. After centrifugation, discard the transparent waste liquid in the lower collection tube.

[0077] (3) After putting the adsorption column back into the collection tube, add the orange-yellow liquid obtained in (2) into the adsorption column CB2 in batches.

[0078] (4) Centrifuge at 12,000 rpm and centrifuge again for 1 min. At this time, the orange-yellow waste liquid enters the collection tube, and the DNA fragment has been adsorbed onto the column.

[0079] (5) Replace the adsorption column and add 500 μL of washing solution PW to the adsorption column.

[0080] (6) The centrifugation speed is 12,000 rpm and the centrifugation time is 1.5 min. PW waste liquid in the collection tube is poured out and the adsorption column is put back.

[0081] (7) Repeat operation steps (6) and (7).

[0082] (8) Place the adsorption column back into the collection tube and centrifuge at 12,000 rpm for 2 min to remove as much of the washing liquid as possible.

[0083] (9) Remove the adsorption column from the collection tube and place it in a clean working environment with a small air blower for 3 minutes to ensure that the alcohol in the adsorption column CB2 is completely dried.

[0084] (10) The eluent EB should be preheated in a water bath at 65-70℃.

[0085] (11) Place the dried adsorption column into a 1.5 mL labeled centrifuge tube, add 35-50 μL of elution EB, place it in a cool place at room temperature for 2-5 min, and centrifuge at 12,000 rpm for 2-3 min to completely centrifuge the liquid and collect the DNA solution.

[0086] (12) Take 2 μL of liquid and use a spectrophotometer ND-100 to determine the DNA concentration.

[0087] 4.1.5. Connection between the target fragment and the vector Take 5×TEAD and establish a ligation system (20 μL). The molar ratio of the target fragment to the vector is approximately 4:1. Place the system in a PCR chamber and set the program to 30℃ for 40 min for ligation.

[0088] Table 7: Connection System Table

[0089] The formulation for 5 × TEAD is: 1 mL 5 × TEAD buffer plus 1 μL T5 exonuclease.

[0090] The formulation of 5×TEAD buffer (10mL) is as follows: 0.5 M Tris-HCl, 50 mm DTT, 50 mm MgCl2, 0.25 g PEG8000, T5 Exonuclease (10 U / mL).

[0091] 4.1.6. Escherichia coli transformation: (1) When the above system is about to end, take out the DH5α competent state prepared in step 2 from the -80℃ freezer. After taking it out of the freezer, quickly insert it into ice and let it melt on the ice for about 6 minutes.

[0092] (2) After the T5 ligase ligation is completed, transfer the product to the thawed competent cells, gently tap the bottom of the EP tube by hand, and then place it on ice for 10 min.

[0093] (3) Heat shock in a water bath at 42℃ for 90 s, then quickly put it back on ice and let it stand for 5 min.

[0094] (4) Spread on LB solid plates containing kanamycin, and after the surface is completely dried in a clean bench, incubate overnight at 37°C.

[0095] 4.1.7. Validation of recombinant plasmid colonies by PCR Single colonies grown from the above plates were picked and transferred to 5 mL of LB liquid medium containing 30 μg / mL kanamycin. The culture was incubated at 37°C for 5 h using a shaker. One μL of the bacterial culture was then taken out (the remaining culture was centrifuged and stored at -20°C) for colony PCR verification. T7 universal primers were used for the upstream primers, and primers for the downstream fragments were used for the corresponding target fragments.

[0096] Table 8: Reaction System Table

[0097] After colony PCR, take 10 μL of the product directly without adding loading buffer for agarose gel electrophoresis and observe the results under UV light.

[0098] 4.1.8. DNA sequencing identification (1) Add anhydrous ethanol to the specified level in the rinsing solution PW. Add the RNase from the kit to P1.

[0099] (2) Take the above colonies and verify the correct colonies by PCR. Use 5 mL of LB liquid medium to culture until the plateau phase.

[0100] (3) Centrifuge the cells using a 1.5 mL centrifuge tube. For the last collection, use a 100 μL pipette to remove the upper layer of culture medium.

[0101] (4) Slowly add 250 μL of solution P1 to the centrifuge tube above, and resuspend the Escherichia coli bacteria at the bottom with a 1 mL pipette or vortex mixer.

[0102] (5) Slowly add 250 μL of solution P2 to the centrifuge tube and gently shake it from side to side a few times to ensure complete lysis of the bacteria.

[0103] (6) Slowly add 350 μL of solution P3 to the centrifuge tube, and immediately gently shake it from side to side several times until it is completely mixed. Place the centrifuge tube in a centrifuge and centrifuge at 12,000 rpm for 10 min at room temperature.

[0104] (7) During the 10 min centrifugation in the previous step, equilibrate the adsorption column: After removing the adsorption column, attach it to the collection tube. After labeling it with a marker, add 500 μL of equilibration solution, centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and then put the adsorption column back in.

[0105] (8) After centrifuging the mixture in (5) to the bottom completely, if not, centrifuge again.

[0106] (9) After high-speed centrifugation, add the supernatant to the pre-equilibrated adsorption column using a suitable pipette according to the label. Do not let the pipette tip touch the milky white precipitate below. Centrifuge at 12,000 rpm for 45 s, discard the waste liquid, and return the adsorption column to its original position.

[0107] (10) After ensuring that all supernatant has passed through the adsorption column, add 600 μL of wash buffer PW, centrifuge at 12,000 rpm for 60 s, empty the liquid in the collection tube, and put the adsorption column back.

[0108] (11) Repeat the above operation (9) steps.

[0109] (12) Remove the remaining alcohol washing solution from the adsorption column completely, then put the washed adsorption column back into the collection tube and centrifuge at 12,000 rpm for 2 min.

[0110] (13) Use a marker to mark the corresponding parameters on the clean centrifuge tube cap, take out the adsorption column and place it in it, open the adsorption column and add 35 μL of elution solution to the center, being careful not to touch the adsorption membrane.

[0111] (14) After placing the adsorption column in a cool place at room temperature for 2-10 min, centrifuge at the highest speed of 12,000 rpm for 2 min and collect the obtained plasmid solution for subsequent use.

[0112] (15) Take an appropriate amount and send it to Xiamen Platinum Biotech Co., Ltd. for sequencing. The remaining amount should be labeled and stored in a -20℃ refrigerator.

[0113] 4.2. Transient transduction of CV296-CD63 plasmid in cells (1) Select target cells HEK293T and replace them with antibiotic-free complete culture medium (DMEM + 10% FBS) 1 h before transfection. (2) Use 1~2 μg of CV296-CD63 plasmid (concentration ≥ 0.5 μg / μL) per well for transfection, and mix the plasmid and transfection reagent at a ratio of plasmid (μg): Lipofectamine = 1:2 (w / v); (3) Prepare a transfection solution by mixing the purified plasmid DNA with the liposome transfection reagent at a mass-to-volume ratio of 1:2 (μg:μL). Dilute the plasmid and liposome separately with serum-free DMEM medium, allow to stand at room temperature for 5 min to equilibrate, and slowly add the liposome dilution to the DNA dilution. Incubate at room temperature in the dark for 18 min, remove the original medium from the culture dish, gently wash the cells with PBS, and then add the transfection complex dropwise along the edge of the culture dish. Use serum-free DMEM for the initial transfection stage (4 hours), and replace it with complete medium containing 10% fetal bovine serum (FBS) after 4 hours. (4) Place in an incubator at 37℃ and 5% CO2 for 24-48 h; (5) 24 h after transfection, the red fluorescence of mCherry was observed by fluorescence microscopy to evaluate the transfection efficiency.

[0114] 4.3. Detection of 2E localization in 293T cells using immunofluorescence co-localization technique (1) Seed 293T cells in a 2 cm confocal culture dish, and after normal transfection for 24 h, discard the culture medium; (2) Gently add 1 mL of PBS, let stand for 30 s and then aspirate. Repeat 3 times (avoid vigorous shaking to prevent cell detachment). Add 500 μL of 4% paraformaldehyde (prepared with PBS) to each dish, fix at room temperature for 10-15 min, then wash 3 times with PBS (1 mL each time, let stand for 30 s). (3) Add 500 μL of 5% BSA (prepared with PBS) and block at room temperature for 30 min. Discard the waste liquid after blocking. (4) Nanobody 2E is diluted 1:100 with PBS or 1% BSA (prepared with PBS) (final volume 30~40μL). It is evenly added to the center of the culture dish and incubated at room temperature for 1 h (to avoid liquid evaporation, it can be placed in a humidifier). (5) Discard the primary antibody and wash three times with PBS (5 min each time). Add fluorescently labeled secondary antibody (1:500-1:1000 dilution) and incubate at room temperature in the dark for 1 h; (6) Discard the secondary antibody and wash 3 times with PBS (5 min each time). Add 1:1000 DAPI (diluted with PBS) and incubate at room temperature in the dark for 5-10 min; (7) Wash with PBS 3 times (5 min each time), and finally add 1 mL of PBS to prevent the sample from drying out; (8) Confocal microscopy imaging: DAPI is excited by a 405 nm laser, and the fluorescence signal of the target protein is detected by a laser of the corresponding wavelength (488 nm green fluorescence, 594 nm red fluorescence). The Z-axis tomography is adjusted to avoid fluorescence quenching, and the imaging is completed.

[0115] 4.4. Immunofluorescence co-localization technique for detecting the localization of CD63 and 2E in 293T-sgCD63 cells CD63-knockout 293T cells were constructed using CRISPR-Cas9 gene editing technology. Immunofluorescence colocalization analysis of CD63 and 2E was performed in the ko cell line 293T-sgCD63 (commercially available, such as from Hwayuan Biotechnology (Shanghai) Co., Ltd.) and the control cell line 293T-sgNC (commercially available, such as from Hwayuan Biotechnology (Shanghai) Co., Ltd.).

[0116] Four treatment conditions were designed for the experiment: transfection with CV296-CD63 plasmid (commercially available, such as from Heyuan Biotechnology (Shanghai) Co., Ltd.) and incubation with 2E nanobodies; transfection with CV296-CD63 plasmid only without incubation with 2E; no plasmid transfection but incubation with 2E nanobodies; and no plasmid transfection and no incubation with 2E nanobodies.

[0117] For detailed steps, refer to 4.2. Transient transduction of CV296-CD63 plasmid in cells and 4.3. Immunofluorescence colocalization technique to detect the localization of CD63 and 2E in 293T cells.

[0118] See results Figure 5-6 .in Figure 5 This is an immunofluorescence co-localization analysis showing the specific binding of nanobody 2E to CD63 protein on the surface of HEK293T cell membranes. Image a shows the IFA identification results of CV296-CD63 transfected HEK293T cells (Blank: CV296 empty vector transfection group). Image b shows the immunofluorescence co-localization analysis of 2E and CD63 in HEK293T cells, with three different fields of view. mCherry (red): mCherry staining of CD63 protein; DAPI (blue): DAPI staining of cell nuclei; FITC (green): conjugated with the antibody. When an antibody carrying FITC specifically binds to a target antigen (such as a specific protein), it emits green fluorescence under blue light excitation, thus indicating the location of the antigen. Merged: This is a composite image generated by superimposing two or more single-channel fluorescence images.

[0119] Figure 6This image shows the results of verifying the specific binding of nanobody 2E to CD63 in 293T cells in Example 4. Image a shows laser confocal imaging of 293T-sgNC cells under four different treatment conditions. The treatment conditions were: transfection with CV296-CD63 plasmid and incubation with 2E nanobody (denoted as CD63-OE NB-2E). Transfecting only the CV296-CD63 plasmid without incubation for 2E (denoted as CD63-OE); undiluted plasmid but incubated for 2E nanobodies (denoted as ctlNB-2E). (b) shows a laser confocal imaging image taken in 293T-sgCD63 cells under the same treatment conditions as above, without transfection with plasmids or incubation of 2E nanobodies (denoted as ctl). Fluorescence channels: DAPI (blue, nucleus); mCherry (red, CD63); FITC (green, 2E nanobodies); Merge (merged image, yellow areas indicate co-localization).

[0120] Nanobody 2E can recognize and bind to CD63 expressed on the surface of 293T cell membranes. Recombinant plasmids were introduced into 293T cells via liposome transfection. Forty-eight hours after transfection, fluorescence microscopy revealed a clear mCherry fluorescence signal in the cell membrane region of the CV296-CD63 transfected group, while no specific fluorescence was observed in the empty vector control group (i.e., Blank) (Figure 5a). This result confirms the successful expression of CV296-CD63 in eukaryotic cells. To verify the specific binding of 2E to CD63, immunofluorescence co-localization analysis was used. His-tagged nanobody 2E was added to the CV296-CD63-expressing 293T cell system to bind to CD63-ECD, followed by specific recognition using FITC-labeled anti-His secondary antibody. Laser confocal microscopy revealed that the green fluorescent signal (2E) and the red fluorescent signal (CD63) exhibited significant spatial co-localization in the cell membrane region, and no non-specific binding signal was observed in the cell nucleus region (Figure 5b).

[0121] To further validate the specific binding of the 2E nanobody to CD63 in 293T cells, CD63-knockout 293T cells were constructed using CRISPR-Cas9 gene editing technology. Immunofluorescence co-localization analysis of CD63 and 2E was performed in the KO cell line 293T-sgCD63 and the control cell line 293T-sgNC. Four treatment conditions were designed: transfection with the CV296-CD63 plasmid followed by incubation with the 2E nanobody (denoted as CD63-OE NB-2E). Transfecting only the CV296-CD63 plasmid without incubation for 2E (denoted as CD63-OE); undiluted plasmid but incubated for 2E nanobodies (denoted as ctl NB-2E). The results showed that in the control group 293T-sgNC cells: no fluorescence signal was observed under the conditions of no plasmid transfection and no 2E nanobody incubation; red fluorescence signal was observed on the cell membrane in the group transfected only with CV296-CD63 plasmid, indicating that CV296-CD63 is normally expressed in the cells; green fluorescence signal was observed on the cell membrane in the group incubated only with 2E nanobody, which may be due to the binding of 2E to endogenously expressed CD63 in the cells; a high degree of colocalization of red and green fluorescence was observed in the group transfected with CV296-CD63 plasmid and incubated with 2E nanobody, indicating that 2E significantly binds to CD63-mCherry on the cell membrane (Figure 6a).

[0122] Notably, the appearance of green fluorescence in the 2E nanobody group was either due to the binding of 2E to endogenously expressed CD63 or a non-specific binding. Therefore, to rigorously verify the binding specificity and investigate the impact of CD63 deficiency on the localization of the 2E nanobody, we simultaneously treated a CD63 gene knockout 293T cell model (CD63-KO) with the same method. The results showed that in the knockout group 293T-sgCD63 cells: no fluorescence signal was observed under conditions of no plasmid transfection and no 2E nanobody incubation; red fluorescence signal was observed on the cell membrane in the group transfected only with the CD63-mCherry plasmid; no fluorescence signal was observed in the group incubated only with the 2E nanobody; and a high degree of co-localization of red and green fluorescence was observed in the group transfected with the CV296-CD63 plasmid and incubated with the 2E nanobody (Figure 6b). By comparing the distribution of green fluorescence signals in 293T-sgCD63 cells and 293T-sgNC cells treated with only 2E antibody, it was determined that in 293T-sgNC cells, 2E antibody binds to endogenously expressed CD63 on the cell membrane; when endogenous CD63 is knocked out, 2E antibody cannot bind. Furthermore, when exogenous CD63 expression is introduced into 293T-sgCD63 cells lacking endogenous CD63, 2E can bind highly specifically to CD63 on the cell membrane. These experimental results confirm the absolute specificity of the binding of the nanobody 2E to CD63.

[0123] In summary, the high specificity of nanobody 2E in recognizing and binding to CD63 lays an experimental foundation for the subsequent development of highly sensitive and specific CD63 detection technologies based on this nanobody. These findings have significant scientific value for advancing CD63-related biological research and its clinical applications.

[0124] 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 without departing from the principles and spirit of the present invention.

Claims

1. A nanobody, characterized in that, It contains complementarity-determining regions CDR1, CDR2, and CDR3, the amino acid sequences of which are shown in SEQ ID NO: 3, 4, and 5, respectively; Preferably, the nanobody has an amino acid sequence as shown in SEQ ID NO:

1.

2. A nucleotide, characterized in that, Its encoding is the nanobody described in claim 1; Preferably, the nucleotide sequence is as shown in SEQ ID NO:

2.

3. An expression carrier, characterized in that, It contains the polynucleotide as described in claim 2.

4. A host cell, characterized in that, It contains the nucleotide as described in claim 2, or the expression vector as described in claim 3.

5. A method for preparing the nanobody of claim 1, characterized in that, include: The host cells of claim 4 are cultured, and the nanobodies are isolated and purified from the culture.

6. The nanobody of claim 1 has the use of binding CD63 protein.

7. A kit for detecting or binding CD63 protein, characterized in that, It contains the nanobody as described in claim 1.

8. Use of the nanobody of claim 1 in the preparation of products for the separation, detection or targeting of exosomes; Preferably, the product is an immunoaffinity chromatography medium, an ELISA detection reagent, an immunofluorescence detection reagent, or an in vitro diagnostic device.

9. A product for separating, detecting, or targeting exosomes, characterized in that, It contains the nanobody as described in claim 1.

10. The product as described in claim 9, characterized in that, The product is an immunoaffinity chromatography medium, ELISA detection reagent, immunofluorescence detection reagent, or in vitro diagnostic equipment.

Images