A mouse monoclonal antibody against lamp1 and use thereof

Abstract

The application discloses an anti-LAMP1 monoclonal antibody or antigen binding fragment thereof and application, including a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region is shown as SEQ ID NO.1, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.2. The anti-LAMP1 monoclonal antibody of the application can be combined with LAMP1 with high affinity and high specificity, realizes specific combination of LAMP1. A specific mouse monoclonal antibody for the cytoplasmic tail of LAMP1 is prepared, and new tools and resources are provided for structural and functional research of LAMP1 protein.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a mouse monoclonal antibody against LAMP1 and its application. Background Technology

[0002] Lysosome-associated membrane protein (LAMP1) is a type I membrane protein, primarily composed of an intraluminal domain, a transmembrane region, and a short cytoplasmic tail. The intraluminal domain can be further divided into two internal homologous domains separated by a hinge region rich in proline, serine, or threonine. The transmembrane region of LAMP1 is located at its C-terminus, forming a single transmembrane domain. This structure anchors LAMP1 to the lysosomal membrane, placing the majority of its protein region (approximately 80%) within the lysosomal lumen, with only the C-terminal tail exposed to the cytoplasm. Furthermore, LAMP1 cDNA has been successfully isolated from cells of humans, mice, rats, and chickens. These different species of LAMP1 proteins exhibit significant conservation in their primary sequence, demonstrating similarities in structural features and biochemical properties.

[0003] LAMP1, a marker protein of the lysosomal membrane, is primarily located in lysosomes and late endosomes, with a small distribution also detectable on the cytoplasmic membrane. In immune cells, LAMP1, or CD107a, is one of the main proteins in the cytolytic granules of natural killer cells (NK cells) and cytotoxic T cells (CTLs), making it crucial in the immune response. LAMP1 expression is not limited to specific cell types but is widely distributed in many other cell types, such as activated platelets, activated lymphocytes, macrophages, epithelial cells, and endothelial cells. Under pathological conditions, the expression and distribution of LAMP1 often change significantly. For example, in various tumors such as colorectal cancer and breast cancer, LAMP1 exhibits high levels of expression on the lysosomal membranes of cancer cells. In lysosomal dysfunction, abnormal distribution of LAMP1 is often accompanied by increased lysosomal volume or rupture, leading to substrate accumulation due to impaired degradation, thereby affecting cell stability. In diseases such as Alzheimer's, LAMP1 expression is increased in neurons containing abnormal aggregations of β-amyloid protein, suggesting its involvement in the elimination of abnormal proteins or lysosomal stress responses.

[0004] The short cytoplasmic tail of LAMP1 consists of 11 amino acid residues. The tail structure of LAMP1 is similar in humans, mice, rats and chickens, showing high conservation. Currently, there are commercially available antibodies targeting this short cytoplasmic tail region, but they are all polyclonal. Therefore, in order to obtain a more stable and specific monoclonal antibody, it is necessary to develop a highly sensitive monoclonal antibody, which provides new tools and resources for the study of the structure and function of LAMP1 protein. Summary of the Invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a monoclonal antibody against LAMP1 or its antigen-binding fragment or a single-chain antibody against LAMP1.

[0006] Another technical problem that this invention aims to solve is to provide a nucleic acid molecule.

[0007] Another technical problem that this invention aims to solve is to provide expression cassettes, recombinant vectors, recombinant cells, or recombinant bacteria.

[0008] Another technical problem to be solved by the present invention is to provide the application of the monoclonal antibody or its antigen-binding fragment, the single-chain antibody against LAMP1, the nucleic acid molecule or the expression cassette, recombinant vector, recombinant cell or recombinant bacteria in the preparation of detection reagents or kits for detecting lysosome-associated membrane protein (LAMP1).

[0009] Another technical problem that this invention aims to solve is to provide a detection reagent or kit for lysosome-associated membrane proteins.

[0010] The final technical problem to be solved by this invention is to provide a method for detecting the presence or level of lysosome-associated membrane proteins in a sample.

[0011] Technical solution: In order to solve the above-mentioned technical problems, the present invention provides a monoclonal antibody against LAMP1 or its antigen-binding fragment, wherein the monoclonal antibody against LAMP1 or its antigen-binding fragment includes a light chain variable region and a heavy chain variable region, wherein the amino acid sequence of the light chain variable region is shown in SEQ ID NO.1 and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.2.

[0012] The heavy chain variable region has amino acid sequences CDR1, CDR2, and CDR3, as shown in Table 7; the light chain variable region has amino acid sequences CDR1, CDR2, and CDR3, as shown in Table 8.

[0013] The nucleotide sequence of the light chain variable region is shown in SEQ ID NO.3, and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.4.

[0014] The monoclonal antibody or its antigen-binding fragment further includes a constant region. Preferably, the constant region is selected from any one of the following: the constant region of IgG, IgA or IgM antibody.

[0015] The present invention also includes a single-chain antibody against LAMP1, the amino acid sequence of which is shown in SEQ ID NO.5, and the corresponding E. coli codon-optimized nucleotide sequence is shown in SEQ ID NO.6.

[0016] In some feasible implementations, the antigen-binding fragment of the monoclonal antibody is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragments, single-chain antibodies, human antibodies, chimeric antibodies, bispecific or multispecific antibodies.

[0017] The present invention also includes a nucleic acid molecule encoding the aforementioned monoclonal antibody or its antigen-binding fragment, or a single-chain antibody against LAMP1. This nucleic acid molecule can be a polynucleotide, and the polynucleotide is not limited to any particular method of production and can be obtained using genetic engineering recombination technology or chemical synthesis methods.

[0018] The present invention also includes expression cassettes, recombinant vectors, recombinant cells or recombinant bacteria, which contain the aforementioned nucleic acid molecules.

[0019] The present invention also includes nucleic acid constructs comprising the polynucleotides described above, and at least one expression regulatory element operatively linked to the polynucleotides.

[0020] The recombinant vector of the present invention can be a cloning vector or an expression vector, for example, it can be a plasmid, a granule, a bacteriophage, etc.

[0021] In some preferred embodiments, the recombinant vector is a recombinant expression vector, preferably a eukaryotic expression vector.

[0022] The recombinant cells described in this invention include a transformed host cell, wherein the transformed cell is a polynucleotide as described above, a nucleic acid construct as described above, or a recombinant vector as described above.

[0023] The host cells include, but are not limited to: prokaryotic cells, such as Escherichia coli cells; eukaryotic cells, such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). The host cells can also be cell lines, such as the 293T cell line. Preferably, the host cells are eukaryotic cells, and more preferably mammalian cells.

[0024] The present invention also includes the application of the monoclonal antibody or its antigen-binding fragment, the single-chain antibody against LAMP1, the nucleic acid molecule or the expression cassette, recombinant vector, recombinant cell or recombinant bacteria in the preparation of a detection reagent or kit for detecting lysosome-associated membrane protein (LAMP1).

[0025] The present invention also includes a detection reagent or kit for lysosome-associated membrane proteins, wherein the detection reagent or kit comprises the monoclonal antibody or its antigen-binding fragment, the nucleic acid molecule or the expression cassette, recombinant vector, recombinant cell or recombinant bacteria.

[0026] The present invention also includes a method for detecting the presence or level of lysosome-associated membrane proteins in a sample, the method comprising using the monoclonal antibody or its antigen-binding fragment, the anti-LAMP1 single-chain antibody, the nucleic acid molecule, the expression cassette, recombinant vector, recombinant cells, or recombinant or the reagents or kits described herein.

[0027] The samples include, but are not limited to, serum or plasma from the subject.

[0028] The general methods for detecting the presence or level of a target antigen in a sample using monoclonal antibodies or their antigen-binding fragments are well known to those skilled in the art. In some preferred embodiments, the detection method may use enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay, chemiluminescent immunoassay, radioimmunoassay, fluorescence immunoassay, immunochromatography, competitive assays, and similar methods.

[0029] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The anti-LAMP1 monoclonal antibody of the present invention can bind to LAMP1 with high affinity and high specificity, achieving specific binding of LAMP1. A specific mouse monoclonal antibody targeting the cytoplasmic tail of LAMP1 has been prepared, providing new tools and resources for the study of the structure and function of the LAMP1 protein. Attached Figure Description

[0030] Figure 1 The image shows the purification results of the monoclonal antibody as detected by SDS-PAGE.

[0031] Figure 2 Western blot analysis of LAMP1 monoclonal antibody;

[0032] Figure 3 IFA analysis for LAMP1 monoclonal antibody;

[0033] Figure 4 Application of LAMP1 monoclonal antibody in newcastle disease models;

[0034] Figure 5 Western blotting analysis of scFv monoclonal antibody. Detailed Implementation

[0035] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0036] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to embodiments. Those skilled in the art can easily understand other advantages and effects of this invention from the content disclosed in this specification. Any modifications or substitutions made to the methods, steps, or conditions of this invention without departing from the spirit and substance of this invention are within the scope of this invention.

[0037] Example 1: Preparation of LAMP1 monoclonal antibody

[0038] This embodiment provides a method for preparing a monoclonal antibody against LAMP1. The preparation method is based on hybridoma technology and specifically includes the following steps:

[0039] 1. Antigen synthesis

[0040] The antigenic peptide LAMP1 (sequence: GRKRSHAGYQTI) was synthesized by Shanghai Qiangyao Biotechnology Co., Ltd. and conjugated with KLH and BSA, respectively. During the synthesis of LAMP1 peptide, a cysteine ​​C residue was added to the N-terminus to label the carrier protein keyhole limpet hemocyanin (KLH). During the synthesis of LAMP1 peptide, the carrier protein bovine serum albumin (BSA) was linked to the N-terminus via a cysteine ​​residue, ultimately yielding synthetic peptides conjugated with KLH and BSA, respectively. These were used for titer detection in animal immune serum and subsequent detection in mouse monoclonal antibody expression supernatant. The sequence of the synthesized LAMP1 peptide is: GRKRSHAGYQTI.

[0041] 2. Animal immunization

[0042] LAMP1 (LAMP1-KLH), a synthetic polypeptide conjugated with KLH, was used as an immunogen. For the initial immunization, an equal volume of Freund's complete adjuvant was mixed 1:1 and thoroughly emulsified, then 50 μg was administered to each BALB / c mouse. Subsequent immunizations were performed using the same method, emulsifying the protein with Freund's incomplete adjuvant, 50 μg per mouse vial, using multiple subcutaneous sites. Each immunization was spaced 2–3 weeks apart. Seven days after the third immunization, tail blood was collected from mice to test the titer. The collected tail blood was incubated at 4°C for 30 minutes. After serum precipitation, it was serially diluted with sterile physiological saline. A blank mouse serum was used as a control, processed in the same way. The maximum serum dilution factor when the OD value of the immunized mouse serum / negative serum OD value was ≥2.1 was the mouse serum titer. Mice with high titers were selected for booster immunization via intraperitoneal injection of 50 μg of antibody and an equal volume (200 μL) of Freund's incomplete adjuvant emulsion. Three days later, the mice were sacrificed for cell fusion.

[0043] 3. Cell fusion

[0044] Blood was collected from the eyes of mice that had undergone booster immunization, and serum was separated as a positive control. Mice were euthanized by cervical dislocation and immersed in 75% ethanol for 10 minutes. The mice were then secured to a dissection table in a biosafety cabinet. Using sterilized forceps, the abdominal skin was lifted, and a small piece of skin was cut upwards from the lower abdomen using sterile scissors. The skin and peritoneum were separated, and other internal organs were carefully dissected. The spleen was then carefully removed and placed in a culture dish containing 20 mL of DMEM culture medium. Using a syringe filled with 20 mL of DMEM culture medium, the syringe was inserted through the top of the spleen, and spleen cells were gently expelled into the culture dish. This process was repeated several times until the spleen no longer changed color. After filtering through a sieve, the spleen cells were obtained. For SP2 / 0 cells and splenocytes, splenocytes were mixed with SP2 / 0 cells at a ratio of 8:1. After mixing by inversion, the mixture was centrifuged at 1000 rpm for 4 min. Under 37°C water bath conditions, the precipitated cells were tapped to distribute them evenly at the bottom of the tube. After standing for 1 min, 1 mL of PEG 1500 was added to the centrifuge tube within 1 min, and the mixture was stood for 1 min. Then, 1 mL of preheated 37°C DMEM culture medium was added along the tube wall within 30 s to terminate the cell fusion reaction. The pipette tip was submerged in the liquid, and 1 mL of DMEM culture medium was added within 1 min. This step was repeated until 20 mL of DMEM culture medium was added. After slowly adding 30 mL of DMEM culture medium, the mixture was centrifuged at 800 rpm for 4 min, and the supernatant was discarded. Then, 50 mL of DMEM culture medium was added, and the mixture was centrifuged at 800 rpm for 4 min, and the supernatant was discarded. HAT culture medium was added, and the cells were gently blown up and transferred to a 96-well cell culture plate (200 μL / well). The cells were then labeled. Indirect ELISA was performed after 7 days.

[0045] 4. Screening of positive hybridoma cells

[0046] Collect the supernatant from hybridoma cells and screen for positive hybridoma cells using indirect ELISA. Select wells with high OD450 nm values ​​and containing only single cell clusters. Discard the culture medium and add 200 μL of HT medium (500 μL HT additive (100×), 5 mL Australian fetal bovine serum, 500 μL penicillin-streptomycin solution, and 44 mL DMEM medium, mix well). Disperse and count the cells, and seed approximately 200 cells onto half a 96-well plate. Passage the remaining cells to 48-well plates for further culture and cryopreservation. After 7 days, identify the single-cloned cells using ELISA. Subcloning is performed again using the same method. After three subcloning processes, select cells with high OD450 nm values. 450 For single cell clusters with high values, clone them again using the method described above. If there are cells with pore OD values... 450 The values ​​were all high, and the pore OD values ​​were free of cell clumps. 450 If the value is not higher than that of the negative control, it is considered a hybridoma cell line that can secrete monoclonal antibodies.

[0047] 5. Preparation of monoclonal antibodies

[0048] The monoclonal cell lines selected in step 4 were injected into the peritoneal cavity of mice for culture. Peritoneal fluid was then extracted and purified. The specific procedure is as follows: 500 μL of Freund's incomplete adjuvant was injected intraperitoneally into the mice. 24 hours later, approximately 1 × 10⁻⁶ cells were collected. 7 Hybridoma cells were injected into the peritoneal cavity of mice, and ascites fluid was collected 7 days later. Antibodies were purified using a pre-packed Protein A+G Agarose (Fast Flow, 1 mL) column from Shanghai Beyotime Biotechnology Co., Ltd. The specific procedures were as follows: Ascites fluid was centrifuged at 10,000 rpm for 10 min, and the supernatant was collected. 20 μL of the supernatant was used to prepare a sample. Binding buffer, neutralization buffer, and elution buffer were filtered through a 0.45 μm filter and set aside. Ascites fluid was diluted twice with binding buffer. 10 mL of binding buffer was drawn into a syringe and attached to the purification column. After removing air bubbles, the stopcock was slowly pushed to remove the stock solution. 10 mL of binding buffer was drawn into the column at a flow rate of 1 mL / min to equilibrate the column. Diluted ascites fluid was drawn into the syringe at a flow rate of 1 mL / min to bind the antibody to the column. 10 mL of binding buffer was drawn into the column to wash away unbound antibodies until the eluent was colorless. 5 mL of elution buffer was drawn into the column to elute the antibody bound to it. The pH of the collected eluent was adjusted to 7.5 using neutralization buffer. Take 20 μL of sample from each tube to prepare a sample for later use, and identify the purified monoclonal antibody.

[0049] Example 2: Identification of LAMP1 monoclonal antibodies

[0050] 1. Identification of monoclonal antibody subclasses

[0051] The subclasses of LAMP1 monoclonal antibodies were detected using an ELISA kit for identifying murine monoclonal antibody IgG classes / subclasses (Suzhou Biotron Immunotherapy Co., Ltd., catalog number BF06001). The results showed that the heavy chain subtype of the monoclonal antibody was IgG1, and the light chain was κ.

[0052] 2. Monoclonal antibody purity determination

[0053] The purified monoclonal antibody was analyzed by SDS-PAGE electrophoresis. Figure 1 As shown, two clear bands appeared near 25kDa and 50kDa. The band at 25kDa represents the antibody light chain, and the band at 50kDa represents the antibody heavy chain. There were no other impurities, and the purity met the expected requirements.

[0054] 3. Monoclonal antibody specificity identification

[0055] (1) ELISA detection:

[0056] a. Coating: LAMP1-BSA peptide and BSA were coated with coating buffer at 5 μg / mL in microplates, 100 μL per well, and incubated overnight at 4℃.

[0057] b. Blocking: After discarding the liquid in the wells, add 200 μL of PBST to wash the plate 4 times, 5 min each time. Then, add 200 μL of 3% FBS to each well and block at 37°C for 2 h.

[0058] c. Primary antibody incubation: Discard the blocking buffer, add 200 μL PBST to wash the plate 3 times, 5 min each time. Dilute the monoclonal antibody prepared in Example 1 to 2 μg / mL with blocking buffer, 100 μL / well, and incubate at 37°C for 1 h;

[0059] d. Secondary antibody incubation: Discard the primary antibody, add 200 μL PBST to wash the plate 5 times, 5 min each time. Add 100 μL mouse anti-His-tag monoclonal antibody (HRP-labeled) (Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number 30404ES60; diluted 1:5000) to each well, and incubate at 37℃ for 1 h;

[0060] e. Color development: Discard the secondary antibody, add 200 μL PBST to wash the plate 5 times, 5 min each time. Add 100 μL TMB single-component colorimetric solution to each well, and incubate at 37℃ in the dark for 10 min. Then add 50 μL stop solution (2 mol / L H2SO4) to each well. Read the absorbance at 450 nm on a microplate reader;

[0061] The experimental results are shown in Table 1. The results show that the monoclonal antibody prepared in Example 1 can specifically bind to LAMP1-BSA.

[0062] Table 1. Antibody Specificity Detected by ELISA

[0063] <![CDATA[OD 450 ]]> 2.855 0.173 0.134

[0064] (2) Western Blot identification

[0065] The purified monoclonal antibody from Example 1 was specifically identified using Western Blot, and the specific steps are as follows:

[0066] 1) Cleaning the glass plate: Wash off the residual adhesive on the glass plate with clean water, and then rinse with ultrapure water.

[0067] 2) Glue pouring and sample loading:

[0068] a. After aligning the glass plate with the electrophoresis tank core, vertically secure it to the holder and add ultrapure water to check for leaks. After 5-10 minutes, if there are no leaks, use filter paper to absorb any remaining water on the glass plate before preparing to pour the gel.

[0069] b. Prepare the top and bottom layers using Novizan's One-Step PAGE Gel Fast Preparation Kit (8%). After adding the accelerator to the bottom layer, pour it directly in without liquid sealing. Next, pour in the top layer and slowly insert the comb to avoid air bubbles forming between the comb and the gel. Finally, let it stand at room temperature for 30 minutes until the gel is completely set before use.

[0070] c. After adding an appropriate amount of electrophoresis buffer, slowly remove the comb. Then begin loading the samples (lane 1 contains 2 μg of LAMP1-BSA; lane 2 contains 2 μg of BSA; lane HeLa contains total HeLa cell protein, with a loading volume of 20 μg; the preparation steps for the total HeLa cell protein are: lysing HeLa cells using RIAP lysis buffer (Shanghai Beyotime Biotechnology Co., Ltd., catalog number: P0013B) to obtain the corresponding total protein). Ensure the electrophoresis buffer at least covers the small glass plate in the inner tank. When using a micropipette to aspirate the sample, try to aspirate close to the wall to avoid aspirating air bubbles. Insert the pipette tip into the well and slowly add the sample, being careful not to add it too quickly to prevent the sample from overflowing from the well.

[0071] d. Electrophoresis: Electrophoresis conditions: 2 hours, 120V. Electrophoresis is terminated when the bromophenol blue indicator band is about 1 cm above the lower edge of the glass plate.

[0072] 3) Transfer membrane

[0073] a. After cutting off the excess gel, arrange the gel and PVDF membrane neatly in the following order: black plate, sponge, filter paper, gel, PVDF membrane, filter paper, sponge, and transparent plate. Ensure there are no air bubbles between the gel and the PVDF membrane.

[0074] b. Place it in a transfer tank for transfer, setting the time to 2.5 hours and the current to 250 mA. During the transfer process, it should always be kept in an ice-water bath to prevent the heat generated during the transfer from affecting the transfer effect.

[0075] 4) Antibody incubation

[0076] a. Blocking: After the transfer is complete, wash the membrane with PBS for 5 min, add 5% skim milk powder and block on a shaker at room temperature for 2 h;

[0077] b. Primary antibody: The monoclonal antibody purified from ascites fluid prepared in Example 1 was used as the primary antibody (final concentration 2 μg / mL), prepared using 5% skim milk powder, and incubated overnight at 4°C. The membrane was then washed with PBST on a shaker for 5 min / wash × 5 times.

[0078] c. Secondary antibody: Dilute the HRP-labeled goat anti-mouse antibody (Shanghai Beyotime Biotechnology Co., Ltd., catalog number A0216; diluted 1:1000) with blocking buffer and block on a shaker at room temperature for 1 h. Then wash the membrane with PBST on a shaker 5 times, 5 min / time.

[0079] 5) Exposure: After developing the color with ECL developer, expose the product.

[0080] Test results as follows Figure 2 As shown, the monoclonal antibody exhibits good reactivity with both LAMP1-BSA and LAMP1 in HeLa cells, and can effectively recognize the target antigen.

[0081] (3) Indirect immunofluorescence of cells

[0082] The purified antibody from Example 1 was specifically identified using indirect immunofluorescence assay. The specific steps are as follows:

[0083] a. One day in advance, add HeLa cell crawling sheets to a 12-well plate and seed the cells. Once the cell confluence reaches 40%-60%, proceed to the next step.

[0084] b. Cell pretreatment: Rinse the cell slides three times with pre-cooled PBS, discard the PBS, place the culture dish with the slides on a pre-cooled iron block at -80℃ for 30 seconds (press firmly with your hand to ensure full contact between the culture dish and the iron block), and then immediately immerse the culture dish with the slides in a 42℃ water bath for 30 seconds.

[0085] c. Fixation: Immediately after cell treatment, fix the cells with 4% paraformaldehyde at room temperature for 15 min, and wash 3 times with PBS.

[0086] Blocking: Block with 10% FBS-1% BSA-0.3M glycine (prepared with PBS) for 1 hour, then wash 3 times with PBS;

[0087] d. Primary antibody: Dilute the ascites purified monoclonal antibody prepared in Example 1 with 10% FBS-1% BSA-PBS (final concentration of 5 μg / ml), 600 μL per well, incubate at room temperature for 1 h, and then wash 5 times with PBS;

[0088] e. Secondary antibody: Dilute FITC-labeled goat anti-mouse secondary antibody (Shanghai Beyotime Biotechnology Co., Ltd., catalog number: A0568; diluted 1:500) with 10% FBS-1% BSA-PBS, incubate at room temperature for 1 h, and then wash 5 times with PBS;

[0089] f. Mounting: Add 1 drop of mounting medium (containing DAPI) to the slide. Use tweezers to pick up the slide and place the cell side onto the mounting medium, preventing air bubbles from forming. Apply clear nail polish evenly around the slide for mounting and allow it to air dry naturally. Store the slide at 4°C and observe it as soon as possible to prevent fluorescence quenching.

[0090] Test results as follows Figure 3 As shown, the monoclonal antibody exhibited good binding affinity to the LAMP1 protein in HeLa cells, producing a specific green fluorescent signal. In contrast, the control group did not show any fluorescent signal.

[0091] Example 3: Acquisition of LAMP1 antibody sequence

[0092] 1. Extraction of total RNA from hybridoma cell lines

[0093] (1) Cell collection: Collect hybridoma cells, take 1×10 7 Collect cells and transfer the collected cells into centrifuge tubes. Centrifuge at 800 rpm for 3 min at room temperature.

[0094] (2) Cell lysis: After centrifugation, gently aspirate the supernatant to prevent cell loss. Add 1 mL of Trizol reagent to the cell line and mix by pipetting to repeat the lysis process. Continue pipetting until no obvious cell clusters are visible. Transfer the cells to EP tubes and lyse them on ice for 5 min.

[0095] (3) Layering: Add 200 μL of chloroform, shake vigorously to mix Trizol and chloroform thoroughly, then let stand on ice for 5 min, centrifuge at 4℃, 12000×g, for 15 min;

[0096] (4) Collecting the aqueous phase: After centrifugation, the solution will separate into three layers: a colorless aqueous phase (upper layer), a white intermediate layer, and a red organic layer (lower layer). Carefully aspirate the upper aqueous phase into a new centrifuge tube, avoiding the aspiration of the intermediate layer or organic phase;

[0097] (5) Isopropanol precipitation: Add an equal volume of pre-cooled isopropanol to the aqueous phase, gently invert and mix, let stand at 4℃ for 10 min; centrifuge at 12000×g at 4℃ for 10 min, and a white precipitate can be obtained after centrifugation.

[0098] (6) Ethanol washing: Discard the supernatant and add 1 mL of 75% ethanol (prepared with RNase-free ddH2O). Gently tap the bottom of the tube to suspend the precipitate, and invert the tube several times. Let it stand at room temperature for 3-5 minutes. Centrifuge at 12000×g, 4℃ for 5 minutes.

[0099] (7) RNA dissolution: After centrifugation, remove the supernatant. After aspirating most of the liquid, perform a short centrifugation to completely remove the liquid from the EP tube. Dry the precipitate in a clean bench at room temperature for 2-5 minutes. After drying the RNA precipitate, add 10 μL of RNase-free water to dissolve the RNA and store it at -80℃.

[0100] 2. Reverse transcription

[0101] (1) RNA template denaturation: Place the reverse transcription kit on ice and premix the sample according to Table 2; after mixing, place the sample at 65℃ for 5 min, then quickly place it on ice to cool, and let it stand on ice for 2 min.

[0102] Table 2 Reverse Transcription Sample System

[0103]

[0104] (2) Genomic DNA removal: Add 4 μL of 4×gDNAwiper Mix to the sample after the previous step, mix well by pipetting, and react at 42℃ for 2 min;

[0105] (3) First-strand cDNA synthesis: First, prepare the cDNA synthesis reaction system. After the previous step reaction, add 2 μL of 10×RT Mix and 2 μL of HiScript II Enzyme Mix to the sample and mix well. Then, carry out the first-strand cDNA synthesis reaction according to the reaction program in Table 3.

[0106] Table 3 PCR reaction procedure

[0107]

[0108] (4) The obtained cDNA product can be directly used for PCR experiments or stored at -20℃ or -70℃.

[0109] 3. Amplification of antibody genes

[0110] Based on the antibody structure, degenerate primers were designed according to the method of Shen Beifen et al. (Shen Beifen, Chen Zhinan, Liu Minpei. Recombinant Antibodies [M]. Recombinant Antibodies, 2005), as shown in Table 4.

[0111] Table 4 Primer sequences

[0112]

[0113] Note: R=A / G, Y=C / T, M=A / C, K=G / T, S=C / G, W=A / T, H=A / C / T, B=C / G / T, V=A / C / G, D=A / G / T, N=A / C / G / T.

[0114] Using the first-strand cDNA as a template, the full-length genes of the heavy and light chains of the antibody were amplified using the degenerate primers described above. The amplification system is shown in Table 5.

[0115] Table 5. PCR system for amplifying antibody heavy and light chain genes.

[0116]

[0117] Perform PCR according to the PCR reaction conditions in Table 6.

[0118] Table 6 PCR reaction parameters for variable region amplification

[0119]

[0120] 4. Analysis of antibody light and heavy chain variable region sequences

[0121] The obtained PCR products were subjected to nucleic acid electrophoresis. After electrophoresis, the remaining PCR products were directly sent for sequencing. After sequencing, the obtained VH and VL antibody sequences were uploaded to the IgBLAST website for amino acid sequence analysis of CDR1, CDR2, and CDR3.

[0122] The sequencing results of the heavy chain variable region and light chain variable region of the monoclonal antibody are as follows:

[0123] SEQ ID NO.1: Light chain variable region amino acid sequence DILETQTPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWAS TRESGVPDRFTGSGSGTDFTLTISSVQTEDLAVYFCKQSYNLRTFGGGTKLEIK

[0124] SEQ ID NO.2: Heavy chain variable region amino acid sequence QVKLQQSGPELKKPGETVKISCKASGYTFTDFSMHWVKQAPGKGLKWMGWINTKTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTTPYFCTRGGADWFAYWGQGTTVTVS

[0125] Both the light chain and heavy chain variable regions of monoclonal antibodies are composed of complementarity-determining regions and framework regions. The complementarity-determining regions are composed of CDR1, CDR2 and CDR3. The antibody heavy chain variable regions are shown in Table 7; the antibody light chain variable regions are shown in Table 8.

[0126] Table 7 Antibody Heavy Chain Variable Region Sequence

[0127]

[0128] Table 8. Antibody light chain variable region sequence

[0129]

[0130] The genes for the light chain variable region and heavy chain variable region of the antibody are as follows:

[0131] SEQ ID NO.3: Light chain variable region gene sequence GACATTCTGGAGACACAGACTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAAGGTCACTATGAGCTGCAAATCCAGTCAGAGTCTGCTCAACAGTAGAACCCGAAAGAATTACTTGGCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGAT CTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGGTGCAGACTGAAGACCTGGCAGTTTTATTTCTGCAAGCAATCTTATAATCTTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA

[0132] SEQ ID NO.4: Heavy chain variable region gene sequence CAGGTGAAGCTGCAGCAGTCAGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTTATAACCTTCACAGACTTTTCAATGCACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACTAAGACTGG TGAGCCAACATATGCAGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAATGAGGACACGACTCCATATTTCTGTACTAGAGGAGGGGCAGACTGGTTTTGCTTACTGGGGCCAAGGGACCACGGTCACCGTCTCC

[0133] Example 4: Preliminary application of LAMP1 antibody in disease detection

[0134] Studies have shown that NDV hydrolyzes sialic acid residues at the ends of the glycan chains of LAMP1 and LAMP2 through the salivary enzyme activity of its HN viral protein. This process leads to the deglycosylation and degradation of LAMP1 and LAMP2 by cathepsin B in the lysosomal lumen. This indicates that the HN protein of NDV plays an important role in regulating the stability and function of these proteins (Reference: YChen, SZ, TLiao, et al. The HN protein of Newcastledisease virus induces cellapoptosis through the induction of lysosomal membrane permeabilization[J]. PLoS Pathogens, 2024, 20(2):e1011981.).

[0135] Therefore, the LAMP1 antibody was used to detect how disease affects the expression level and glycosylation status of this protein. The specific steps are as follows:

[0136] (1) Cell preparation

[0137] Cells were seeded the day before transfection, treated with trypsin, and counted. It was essential to ensure that the HeLa cells used were in logarithmic growth phase and in good health. In 24-well plates, 0.5 mL of complete culture medium was added to each well, and 4 × 10⁶ cells were seeded. 4We use 10 HeLa cells to ensure that the cell density reaches 50%-80% confluency on the day of transfection.

[0138] (2) Preparation of transfection complex

[0139] a. Dilution of plasmid DNA: Add 0.25 μg of NDV Herts / 33-HN plasmid DNA (a gift from Professor Yu Chen, Y Chen, SZhu, T Liao, et al. The HN protein of Newcastledisease virus induces cellapoptosis through the induction of lysosomal membrane permeabilization[J]. PLoS Pathogens, 2024, 20(2):e1011981.) to 100 μL I. Add serum-reduced medium (Thermo Fisher Scientific (China) Co., Ltd., catalog number: 31985070) and gently stir until well mixed.

[0140] b. Dilution of transfection reagent: Add 1 μL of the above DNA solution. After gently mixing with LTX reagent, incubate at room temperature for about 25 minutes to allow the DNA to form a complex with the transfection reagent.

[0141] (3) Cell transfection

[0142] a. Remove old culture medium: Discard the culture medium from the wells and use... Wash twice with serum-reduced medium (Thermo Fisher Scientific, catalog number: 31985070);

[0143] b. Add the complex: Add 100 μL of the prepared DNA-transfection reagent complex directly to each well, and then gently shake the culture plate back and forth to ensure that it is mixed evenly.

[0144] (4) Culture: Incubate the cells in a 37°C, 5% CO2 incubator for 18 hours.

[0145] (5) Western Blot

[0146] Total cellular protein was extracted from transfected cells using lysis buffer. Simultaneously, total cellular protein from cells transfected with the empty vector plasmid was extracted as a control. These two proteins were used as antigens, and purified monoclonal antibody from ascites fluid was used as the primary antibody for the experiment. Detailed procedures are described in the Western Blot section of Example 2. Results are shown below. Figure 4We extracted total protein from HeLa cells transfected with the NDV Herts / 33-HN plasmid, and also extracted total protein from HeLa cells transfected with an empty vector plasmid as a control. The results showed that after transfection with the NDV Herts / 33-HN plasmid, LAMP1 expression decreased, and the cells were also deglycosylated to some extent, leading to a decrease in molecular weight.

[0147] Example 5: Construction and application of anti-LAMP1 scFv sequences

[0148] 1. Obtain scFv fragments

[0149] The antibody heavy chain variable region and light chain variable region were linked together using linker (G4S)4 (antibody amino acid sequence in Example 3) to obtain the amino acid sequence of the scFv fragment (SEQ ID No. 5):

[0150] QVKLQQSGPELKKPGETVKISCKASGYTFTDFSMHWVKQAPGKGLKWMGWINTKTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTTPYFCTRGGADWFAYWGQGTTVTVSGGGGS GGGGSGGGGSDILETQTPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQTEDLAVYFCKQSYNLRTFGGGTKLEIK

[0151] The amino acid sequence was commissioned to Sangon Biotech (Shanghai) Co., Ltd. for codon optimization in E. coli, and a pET-28a plasmid containing the optimized scFv (containing a His tag) was constructed, followed by scFv expression.

[0152] Optimized scFv sequence (SEQ ID No. 6):

[0153] CAGGTTAAACTGCAGCAGTCTGGTCCGGAACTGAAAAAACCGGGTGAAACCGTTA

[0154] AAATCAGCTGTAAAGCATCTGGTTATAACCTTCACCGATTTTAGCATGCATTGGGTTA

[0155] AACAGGCTCCGGGTAAAGGTCTGAAATGGATGGGTTGGATCAACACCAAAACCGG

[0156] TGAACCGACCTATGCTGATGATTTCAAAGGTCGTTTCGCTTTCTCTCTGGAAACCCA

[0157] GCGCGTCTACCGCATATCTGCAGATCAACAACCTGAAAAACGAAGATACCCACCCC

[0158] GTATTTCTGTACCCGTGGTGGTGCTGATTGGTTTGCGTACTGGGGTCAGGGTACCA

[0159] CCGTTACCGTTAGC GGCGGTGGTGGTAGCGGTGGTGGTGGTAGCGGTGGTGGTGG

[0160] TTCT GATATCCTGGAAACCCAGACTCCGTCTAGCCTGGCGGTTTCTGCTGGTGAAA

[0161] AAGTTACCATGAGCTGTAAAAGCAGCCAGAGCCTGCTGAACTCCCGTACCCGTAA

[0162] AAACTATCTGGCTTGGTATCAGCAGAAACCGGGTCAGTCTCCGAAACTGCTGATCT

[0163] ACTGGGCGAGCACTCGTGAATCTGGTGTTCCGGATCGTTTCACCGGTTCTGGTTCT

[0164] GGTACCGATTTCACCCTGACCATCTCTAGCGTTCAGACCGAAGATCTGGCGGTTTA

[0165] TTTCTGTAAACAGTCTTATAACCTGCGTACCTTCGGTGGTGGTACCAAACTGGAAA

[0166] TTAAA

[0167] Note: The underlined part is the linker part.

[0168] 2. Purification of scFv

[0169] scFv was purified using the His-tagged protein purification kit (denaturing formulation) (product number: P2229S) from Shanghai Beyotime Biotechnology Co., Ltd.

[0170] 3. SCFv specificity verification

[0171] (1) ELISA

[0172] a. Coating: The LAMP1-BSA peptide and BSA synthesized in Example 1 were coated with coating buffer at 5 μg / mL in microplates, 100 μL per well, and incubated overnight at 4°C.

[0173] b. Blocking: After discarding the liquid in the wells, add 200 μL of PBST to wash the plate 4 times, 5 min each time. Then, add 200 μL of 3% FBS to each well and block at 37°C for 2 h.

[0174] c. Primary antibody incubation: Discard the blocking buffer, add 200 μL PBST to wash the plate 3 times, 5 min each time. Dilute the scfv antibody prepared in this example to 5 μg / mL with blocking buffer, 100 μL / well, and incubate at 37°C for 1 h;

[0175] d. Secondary antibody incubation: Discard the primary antibody, add 200 μL PBST to wash the plate 5 times, 5 min each time. Add 100 μL mouse anti-His-tag monoclonal antibody (HRP-labeled) (Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number 30404ES60; diluted 1:1250) to each well, and incubate at 37℃ for 1 h;

[0176] e. Color development: Discard the secondary antibody, add 200 μL PBST to wash the plate 5 times, 5 min each time. Add 100 μL TMB single-component colorimetric solution to each well, and incubate at 37℃ in the dark for 10 min. Then add 50 μL stop solution (2 mol / L H2SO4) to each well. Read the absorbance at 450 nm on a microplate reader;

[0177] The experimental results are shown in Table 9. The results show that scFv can specifically bind to LAMP1-BSA.

[0178] Table 9. ELISA Detection of scFv Specificity

[0179]

[0180] (2) Western Blot

[0181] The experimental procedures are described in Example 2 (Western Blot), and the detection results are as follows: Figure 5 As shown in the figure (1: BSA; 2: LAMP1-BSA; 3: HeLa cell total protein), the scFv antibody showed good reactivity with both LAMP1-BSA and LAMP1 in HeLa cells, and was able to effectively recognize the target antigen.

Claims

1. A monoclonal antibody against LAMP1 or its antigen-binding fragment, characterized in that, The anti-LAMP1 monoclonal antibody or its antigen-binding fragment includes a light chain variable region and a heavy chain variable region, the amino acid sequence of which is shown in SEQ ID NO.1 and the amino acid sequence of which is shown in SEQ ID NO.

2.

2. The monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that, The nucleotide sequence of the light chain variable region is shown in SEQ ID NO.3, and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.

4.

3. The monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that, The monoclonal antibody or its antigen-binding fragment also includes a constant region.

4. The monoclonal antibody or its antigen-binding fragment according to claim 3, characterized in that, The constant region is selected from any one of the following: the constant region of IgG, IgA or IgM antibodies.

5. A single-chain antibody against LAMP1, characterized in that, The amino acid sequence of the single-chain antibody is shown in SEQ ID NO.5, and the corresponding E. coli codon-optimized nucleotide sequence is shown in SEQ ID NO.

6.

6. A nucleic acid molecule, characterized in that, It encodes the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 4, or the anti-LAMP1 single-chain antibody as described in claim 5.

7. An expression cassette, a recombinant vector, a recombinant cell, or a recombinant bacterium, characterized in that, It contains the nucleic acid molecule as described in claim 6.

8. The use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the single-chain antibody against LAMP1 according to claim 5, the nucleic acid molecule according to claim 6, or the expression cassette, recombinant vector, recombinant cell or recombinant bacteria according to claim 7 in the preparation of a detection reagent or kit for detecting the lysosome-associated membrane protein LAMP1.

9. A reagent or kit for detecting the lysosome-associated membrane protein LAMP1, characterized in that, The detection reagent or kit includes the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 4, the anti-LAMP1 single-chain antibody as described in claim 5, the nucleic acid molecule as described in claim 6, or the expression cassette, recombinant vector, recombinant cell, or recombinant bacteria as described in claim 7.

10. A method for detecting the presence or level of lysosome-associated membrane protein LAMP1 in a sample in vitro, characterized in that, The method includes using the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 4, the anti-LAMP1 single-chain antibody as described in claim 5, the nucleic acid molecule as described in claim 6, the expression cassette as described in claim 7, the recombinant vector, the recombinant cell, or the reagent or kit as described in claim 9, and the method is for the purpose of non-disease diagnosis and treatment.

11. The method according to claim 10, characterized in that, The samples include serum or plasma from the subject.

Images