Visualization and enrichment extraction preparation for lysosome and method thereof

Abstract

The invention discloses a preparation for visual and enrichment extraction of lysosome and a method thereof. According to the invention, a part of transmembrane regions in TMEM192 are designed as lysosome characteristic targeting elements for the first time, so that cell proteome disturbance caused by overexpression of complete functional proteins is avoided. Secondly, the invention also designs a fluorescent protein and an enrichment label based on the nano antibody, so that accurate extraction and cytological imaging of the lysosome are realized. The technology can be used for extracting and imaging the lysosome of a target body, can be combined with other omics technologies to realize systematic research on the lysosome, and has an important application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a preparation and a method for lysosome visualization and enrichment extraction. Background Art

[0002] Lysosomes, as key organelles in cells responsible for biomaterial recycling and metabolic signal transmission, are closely linked to numerous human diseases [1]. However, due to their extremely small size within the cell, the precise identification of lysosomal molecular components has always been a difficult task. Although existing lysosome purification technologies have revealed many mysteries of lysosomal biology, these methods are often time-consuming and labor-intensive, and are prone to the loss of metabolites and adjacent proteins, limiting in-depth research.

[0003] Previous studies have developed an immunoisolation method based on the lysosomal protein TMEM192, LysoIP, which can rapidly isolate intact lysosomes, enabling subsequent multi-omics measurements. However, this method also carries potential risks: overexpressing tagged TMEM192 proteins in cells may interfere with normal cellular function. Although the specific function of TMEM192 is not yet fully understood, studies have indicated that it is closely related to the autophagy and apoptosis processes of HepG2 cells [2,3]. This suggests that overexpression of exogenous intact proteins may disrupt the body's proteome balance, thereby affecting the accuracy of research results.

[0004] [1]NYPark,DHCho.The TBK1-SCF-TMEM192-TAX1BP1 axis: a novel regulatory mechanism for lysophagy,Autophagy 10.1080 / 15548627.2025.2479669(2025), https: / / doi.org / 10.1080 / 15548627.2025.2479669

[0005] [2] Z. Liu, YJLv, YPSong,

[0006] [3] TL Nguyen, J. Schneppenheim, S. Rudnik, R. Lüllmann-Rauch, C. Bernreuther, I. Hermans-Borgmeyer, M. Glatzel, P. Saftig, B. Functional characterization of the lysosomal membrane protein TMEM192 in mice,Oncotarget 8(2017)43635-43652,https: / / doi.org / 10.18632 / oncotarget.17514 Summary of the Invention

[0007] To circumvent this risk, the present invention innovatively designed a strategy for modifying the N-terminus and C-terminus of the human TMEM192 protein, selecting a sequence fragment that can specifically localize to lysosomes. Overexpression of this TMEM192 variant, while retaining its lysosomal localization function, weakens or even loses the original biological activity of TMEM192, thereby achieving effective separation and enrichment of lysosomes without disturbing the original state of the cells. On this basis, we cleverly integrated ALFA nanobody technology and combined this sequence with the ALFA tag [H. M. Kilisch, M. Martínez-Carranza, S. Sograte-Idrissi, A. Rajavel, T. Schlichthaerle, N. Engels, R. Jungmann, P. Stenmark, F. Opazo, S. Frey. The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications, Nature Communications 10(2019), https: / / doi.org / ARTN440310.1038 / s41467-019-12301-7].

[0008] The present invention also constructs a nanobody targeting ALFA and fuses it with a fluorescent protein and a His tag to visualize and enrich lysosomes. The co-expression of these two systems in cells provides a novel lysosome enrichment and visualization system for the present invention—the Lysosome Enrichment and Imaging System (LysoEI).

[0009] LysoEI not only integrates lysosome enrichment and visualization capabilities, but also, with its efficient workflow and seamless compatibility with multi-omics analyses, provides a powerful tool for uncovering the multiple roles of lysosomes in physiology and disease. This lysosome enrichment and visualization system undoubtedly demonstrates broad application prospects and enormous potential in the field of lysosomal research.

[0010] The present invention is achieved through the following technical solutions:

[0011] The present invention provides a method for lysosome visualization and enrichment extraction, comprising the following steps:

[0012] (1) constructing a marker plasmid for overexpressing ΔTMEM192 protein; the ΔTMEM192 protein is a truncation of amino acids 19-267 of the wild-type TMEM192 protein; the marker plasmid also contains a sequence for expressing an ALFA tag;

[0013] (2) constructing a recognition plasmid; the recognition plasmid contains an ALFA tag antibody gene sequence, a fluorescent protein sequence, and a His tag sequence;

[0014] (3) Introducing the constructed marker plasmid and recognition plasmid into the target cells; culturing, dyeing, and visualizing the lysosomes under a confocal microscope; or, enriching and extracting the lysosomes through the affinity interaction between the His tag on the lysosome and the nickel strain.

[0015] Preferably, the sequence of the ΔTMEM192 protein is specifically the protein sequence shown in SEQ ID No. 1.

[0016] Preferably, the marker plasmid in step (1) comprises a sequence for expressing the protein as described in SEQ ID No. 2; and the identification plasmid in step (2) comprises a sequence for expressing the protein as described in SEQ ID No. 5.

[0017] Preferably, the fluorescent protein sequence is the red fluorescent protein mScarlet3.

[0018] Preferably, the target cells in step (3) are HEK293 cells.

[0019] The present invention also provides a preparation for lysosome visualization and enrichment extraction, comprising at least the following two plasmids;

[0020] (1) A marker plasmid for overexpressing ΔTMEM192 protein; the ΔTMEM192 protein is a truncation of amino acids 19-267 of the wild-type TMEM192 protein; the marker plasmid also contains a sequence for expressing an ALFA tag.

[0021] (2) Identification plasmid; the identification plasmid contains ALFA tag antibody gene sequence, fluorescent protein sequence and His tag sequence.

[0022] Preferably, the sequence of the ΔTMEM192 protein is specifically the protein sequence shown in SEQ ID No. 1; and the fluorescent protein sequence is the red fluorescent protein mScarlet3.

[0023] Preferably, the marker plasmid comprises a sequence for expressing the protein as set forth in SEQ ID No. 2; and the recognition plasmid comprises a sequence for expressing the protein as set forth in SEQ ID No. 5.

[0024] The present invention provides the use of ΔTMEM192 protein in preparing a preparation for lysosome visualization and enrichment extraction; the sequence of the ΔTMEM192 protein is specifically the protein sequence shown in SEQ ID No. 1.

[0025] The present invention has but is not limited to the following beneficial effects:

[0026] The present invention is designed to address the visualization and extraction of lysosomes. The traditional approach is to exogenously overexpress the tagged lysosomal membrane protein ΔTMEM192 and then enrich ΔTMEM192. Because overexpression of the complete exogenous protein may change the original cellular protein composition, this time, by comparing different segments of TMEM192, the best-positioned truncation is selected for further optimization. This variant will not affect endogenous lysosomes. Combined with the co-expression of the ALFA tag and anti-ALFA nanoantibody, the enrichment and extraction of lysosomes are achieved.

[0027] This study, for the first time, engineered a portion of the transmembrane region of TMEM192 as a lysosomal-specific targeting element, avoiding the perturbation of the cellular proteome caused by overexpression of a fully functional protein. Furthermore, we designed a nanobody-based fluorescent protein and enrichment tag to enable precise extraction and cytological imaging of lysosomes. This technology can be used to extract and image lysosomes in target organisms and can be combined with other omics techniques to enable systematic study of lysosomes, holding significant potential for application.

[0028] Replacing the original wild-type TMEM192 protein with ΔTMEM192 protein does not change the original cell metabolism. Specifically, overexpression of wild-type TMEM192 will lead to a significant increase in the number of lysosomes in the cell, changing the original metabolic background of the cell. Overexpression of the ΔTMEM192 protein provided by the present invention can achieve the same recognition labeling without significantly increasing the number of lysosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to clearly illustrate the specific implementation methods of the present invention and certain detection technologies used in the experiments, the implementation methods and the technologies used will be described below, mainly in the form of drawings.

[0030] Figure 1 Schematic diagram of the principle of the lysosome enrichment and visualization system of the present invention.

[0031] Figure 2 This is a diagram showing the distribution of lysosomes in cells where the LysoSE system appears.

[0032] Figure 3 Comparison of the effects of ΔTMEM192 (127-267aa) and wild-type TMEM192 on lysosomes in cells.

[0033] Figure 4 Diagram showing the effect of LysoSE system enriching lysosomes.

[0034] Specific implementation methods

[0035] The specific implementation methods of the present invention are explained with the aid of examples. Except that the technology used for detection does not impose any form of limitation on the present invention, some schemes in the described embodiments are part of the embodiments of the invention. The embodiments obtained by ordinary technical operators in this field without creative results are all within the scope of protection of the present invention.

[0036] Example 1 Experiment and Methods

[0037] 1. Construction of Stable Cell Lines with Lentivirus

[0038] 1. Plating 293T cells: Plating 293T cells in a six-well plate so that each well contains approximately 60% of the cells;

[0039] 2. 2 μg of each of the plasmids (A) and (B) and the packaging plasmid (purchased from Quanyang Biotechnology Co., Ltd.) were transfected into the cell dish using 4 μL / tube of Lip3000 (Sigma);

[0040] 3. 48 hours after transfection, remove 3 mL of cell supernatant and centrifuge at 230 g to remove cell debris;

[0041] 4. Infect target HEK293 cells: Filter the virus through a 0.45 μM filter to remove cell debris, then add it to the target cells;

[0042] 5. Resistance screening: Two days after infection, the medium was changed, and HEK293 cell lines were screened with 1 μg / mL puromycin and 5 μg / mL blasticidin until there was no significant change in cell number, after which the drugs were withdrawn.

[0043] 6. Western blotting to detect expression effects: 3 days after drug withdrawal, harvest cells and perform Western blotting to verify expression, lysosome extraction, and cell imaging.

[0044] 7. Freeze the successfully constructed cell line and store it in a -80℃ liquid nitrogen tank.

[0045] 2. Lysosome extraction experiment based on LysoEI:

[0046] 1. Pre-cool the Dounce homogenizer on ice. Keep the sample at a low temperature throughout the entire process.

[0047] 2. When the cells grow to 90% density, digest them, collect the cells, wash them twice with cold PBS, and aspirate the supernatant.

[0048] 3. Add 500 μl of cold solution A of the kit to resuspend the cells, add appropriate amount of protease inhibitors, and let it stand on ice for 10 minutes.

[0049] 4. Homogenize with a Dounce homogenizer for 30-40 strokes, rinse the homogenizer with 500 μl of solution A, and combine and collect the homogenate.

[0050] 5. Centrifuge the homogenate at 4°C, 500×g for 5 minutes. Discard the precipitate and collect the supernatant. (If the liquid is clear, this step is not necessary.)

[0051] 6. Determine the protein concentration of the sample. Pipette a certain amount of sample into a new centrifuge tube for protein quantification and fill up the volume with Biotin A solution. The remaining sample is used as whole cell lysate for subsequent testing.

[0052] 7. Take an appropriate amount of mixed nickel beads, centrifuge at 4°C (1000g × 10s), discard the storage solution, add non-denaturing lysis buffer to the gel and mix well to balance the gel, centrifuge at 4°C (1000g × 10s), discard the supernatant, repeat the balance 1-2 times, and discard the supernatant. (Activate beads)

[0053] 8. Add an equal volume of the quantified sample to the above gel and mix thoroughly. Slowly shake on a Ferris wheel shaker at 4°C for 60 minutes.

[0054] The gel was precipitated by centrifugation at 9.4°C (250 g × 10 s), and the supernatant was kept as a sample of cytoplasmic protein for subsequent detection.

[0055] 10. Add appropriate amount of PBS to resuspend the gel, centrifuge at 4°C (1000g×10s), and take the supernatant for subsequent detection.

[0056] 11. Repeat step 10 and wash once more, for a total of 3 washes.

[0057] 12. Centrifuge for the final time at 4°C (1000g x 10s) and discard the supernatant (use a small gun to rinse dry, avoiding touching the precipitate). The precipitate is the purified nickel-containing lysosome strain.

[0058] 13. Prepare Cell lysate (CST) lysis buffer and 5× loading buffer in the appropriate proportions and add equal volumes to each sample.

[0059] 14. Equal volumes of the above components were loaded for immunoblotting to detect His, Lamp1, and TEM192 protein markers.

[0060] 3. Western Blotting

[0061] 1. Select and prepare a separation gel of appropriate solubility according to the molecular weight of the protein to be detected;

[0062] 2. Take a certain amount of sample after measuring the solubility according to the experimental purpose, add 5× loading tube in a certain proportion, boil in boiling water for 5 minutes, place on ice for 2 minutes, and centrifuge for 10 seconds to allow the liquid on the side wall to return to the bottom of the EP tube. Prepare for sample loading;

[0063] 3. Install the electrophoresis apparatus, add Running Buffer, load the sample and pre-set protein marker;

[0064] 4. After loading the sample, use a voltage of 70-80V for the stacking gel and 80-120V for the separation gel until the bromophenol blue band approaches the end of the electrophoresis. Cut the PVDF membrane to the appropriate size according to the size of the gel strip. 5. Activate the PVDF membrane by soaking it in methanol until it changes color. Arrange it in the order of filter paper, gel, PVDF membrane, and filter paper. Connect the power supply and transfer the membrane on ice at a constant current of 235mA for 75-90 minutes.

[0065] 6. After transfer, remove the PVDF membrane, rinse with TBST to remove the remaining transfer buffer, remove the transfer buffer, and then block with 5% skim milk at room temperature for 2 hours;

[0066] 7. After blocking, rinse with TBST to remove residual milk, cut the membrane according to the protein marker, add the corresponding primary antibody, and incubate at 4°C overnight (12-16 hours);

[0067] 8. Recover the primary antibody, wash the membrane with TBST for 30 minutes, change the solution every ten minutes, add the corresponding secondary antibody, and incubate at room temperature for 2 hours;

[0068] 9. Recover the secondary antibody and wash the membrane with TBST for 30 minutes, changing the solution every ten minutes;

[0069] 10. Reasonably control the exposure time and development, crop the image with Photoshop, and calculate the grayscale value with ImageJ.

[0070] 4. Flow cytometry detection of lysosomes

[0071] 1. Transfect the plated cells with different plasmids. After stimulation, discard the culture medium in the cells. Digest the cells with trypsin, wash the cells 1-2 times with PBS solution, centrifuge at 300g for 2 minutes, and discard the supernatant.

[0072] 2. Dilute the lysosomal dye LysoTracker-Green (Biyuntian C1047S) with PBS solution at a ratio of 1:5000, add an appropriate amount of the diluted dye to the cell pellet and resuspend it (keep the sample in the dark from then on), place it in a 37°C incubator and incubate in the dark for 30 minutes, inverting and mixing every 5-10 minutes to ensure full contact between the dye and the cells.

[0073] 3. After staining, centrifuge at 300g for 2 minutes to remove the supernatant dye, wash the cells three times with PBS solution, and be gentle when pipetting the cells. After centrifugation, discard the supernatant.

[0074] 4. After washing, resuspend the cells in 500 μL PBS, filter the cell suspension through a mesh and transfer it to a flow cytometer. Set the FITC channel of the flow cytometer to detect the lysosomal level of the sample.

[0075] 5. Lysosome Imaging Detection Based on LysoEI System

[0076] LysoEI-expressing HEK293 cells were plated onto confocal microplates at a density of 15,000 cells per dish. The next day, after the cells had adhered, the culture medium was removed with a pipette. The cells were rinsed three times with PBS, fixed with 4% paraformaldehyde for 30 minutes, and then rinsed three times with PBS. The cells were permeabilized with 0.5% Triton-100 for 10 minutes. After rinsing three times with TBST, the cells were stained with DAPI at a dilution of 1:3000 for 3 minutes. Protect from light during staining. The cells were rinsed five times with TBST and then stored in a dark place. Cell fluorescence was then imaged using a confocal microscope, and images were analyzed using ZEN 2.

[0077] 6. Antibody Name and Reagents

[0078]

[0079] Example 2 Experimental Technical Route Design and Results

[0080] LysoEI (Lysosome Enrichment and Imaging System) Technical Principle

[0081] Figure 1 Figure 2 shows the LysoEI system, which consists of two parts: (A) ΔTMEM192(19-267)-ALFA and (D) pCDH-CMV-MCS-EF1-Blast-mScarlet-NbALFA-10His. When cells express these two plasmids, the nanobody-ALFA in (D) can recognize the ALFA sequence epitope on the lysosome. The mScarlet3+10His fused to the nanobody-ALFA can tightly bind to the lysosome, enabling cellular visualization of the lysosome. At the same time, the 10His tag attached to it allows the enrichment of lysosomes by nickel column purification.

[0082] Selection of ΔTMEM192 gene sequence

[0083] Based on the above ideas, we need to further determine which segment of the TMEM192 protein retains its lysosomal localization ability. This protein has four transmembrane regions, and the N-terminal -19-24 sites are essential for its membrane localization, so we retained this region. Near site 126 is the second cytoplasmic region of TMEM192, and we speculated whether the two transmembrane domains generated by truncation here can still be lysosomal. Therefore, three TMEM192 truncation variants were constructed: (A) ΔTMEM192 (19-267aa), (B) ΔTMEM192 (19-126aa), and (C) ΔTMEM192 (127-267aa). Flag and ALFA peptides were added to the ends for identification and labeling. The localization of the three truncation variants in lysosomes was further observed.

[0084] To achieve visualization, we constructed the (D)Nb-mScarlet-10His plasmid, which can express a nanobody targeting the ALFA peptide. The nanobody is fused with the red fluorescent protein mScarlet3 and the 10His tag, which can be used for visualization and enrichment.

[0085] The specific plasmid construction is as follows:

[0086] Recombinant plasmids containing different truncations of pCDH-Puro-ΔTMEM192-ALFA were constructed on the pCDH-CMV-MCS-EF1-puro (Miaoling, Cat.#P21108) backbone. Recombinant plasmids containing pCDH-CMV-MCS-EF1-Blast-mScarlet-NanobodyALFA-10His were constructed on the pCDH-CMV-MCS-EF1-Blast (Miaoling, Cat.#P14038) backbone. All of these plasmids were synthesized by Guangzhou Ruibo Biotechnology Co., Ltd.

[0087] (A) The specific protein sequence of pCDH-Puro-ΔTMEM192(19-267)-flag-ALFA is as follows: the underlined part ( EFDYKDDDDKP ) is the flag tag, the bold part is the ALFA tag sequence; the preceding portion is the ΔTMEM192 protein sequence. Specifically, the protein sequence of ΔTMEM192 (19-267) is as shown in SEQ ID No. 1; the sequence of the entire overexpression plasmid is as shown in SEQ ID No. 2, specifically as follows:

[0088]

[0089] (B) The specific protein sequence of pCDH-Puro-ΔTMEM192(19-126)-flag-ALFA is as follows: the underlined part ( EFDYKDDDDKP ) is the flag tag, the bold part For ALFA

[0090] Tag sequence; the front part is the ΔTMEM192 protein sequence; the sequence of the entire overexpression plasmid is shown in SEQ ID No. 3, as follows:

[0091]

[0092] (C) The specific protein sequence of pCDH-Puro-ΔTMEM192(127-267)-flag-ALFA is as follows: the underlined part ( EFDYKDDDDKP ) is the flag tag, the bold part is the ALFA tag sequence; the preceding portion is the ΔTMEM192 protein sequence; the sequence of the entire overexpression plasmid is shown in SEQ ID No. 4, as follows:

[0093]

[0094] (D) The specific protein sequence of pCDH-CMV-MCS-EF1-Blast-mScarlet-NbALFA-10His is as follows: the bold and underlined portion is the 10His tag sequence: the sequence of the entire overexpression plasmid is shown in SEQ ID No. 5, as follows:

[0095]

[0096] (E) The specific protein sequence of pCDH-Puro-TMEM192-flag is as follows: This plasmid is a plasmid of the wild-type TMEM192 gene, wherein the underlined portion is the flag tag; the sequence of the entire overexpression plasmid is shown in SEQ ID No. 6, which is as follows:

[0097] MAAGGRMEDGSLDITQSIEDDPLLDAQLLPHHSLQAHFRPRFHPLPTVIIVNLLWFIHLVFVVLAFLTGVLCSYPNPNEDKCPGNYTNPLKVQTVIILGKVILWILHLLLECYIQYHHSKIRNRGYNLIYRSTRH LKRLALMIQSSGNTVLLLILCMQHSFPEPGRLYLDLILAILALELICSLICLLIYTVKIRRFNKAKPEPDILEEEKIYAYPSNITSETGFRTISSLEEIVEKQGDTIEYLKRHNALLSKRLLALTSSDLGCQPSRT EFDYKDDDDKP

[0098] Only the ΔTMEM192 (19-267aa) truncated form can localize within lysosomes

[0099] like Figure 2As shown in the figure, based on the above ideas, we constructed a plasmid stably expressing plasmid (D) mScarlat3-nanobody-ALFA-10His in HEK293 cells, and on this basis, overexpressed (A) ΔTMEM192(19-267aa)-ALFA, (B) ΔTMEM192(19-126aa)-ALFA, and (C) ΔTMEM192(127-267aa)-ALFA. At the same time, two control groups overexpressing (A) or (D) alone were set up. As can be seen from the figure, cells overexpressing (A) TMEM192-ALFA alone did not emit light, while cells overexpressing (D) Nb-mScarlet-10His alone emitted red light and showed uniform distribution in the cells, without cell specificity. When (A) + (D) were expressed simultaneously, the cells showed significant red spots, which are consistent with the characteristics of lysosomal localization. In addition, simultaneous overexpression of (B)+(D) or (C)+(D) failed to show lysosomal localization ( Figure 2 ).

[0100] Overexpression of TMEM192 resulted in a significant increase in the number of lysosomes in cells, whereas cells overexpressing ΔTMEM192 (19-267aa) had normal lysosome numbers.

[0101] like Figure 3 As shown in the figure, based on the above results, we found that the (A)ΔTMEM192(127-267aa) variant has significant lysosomal localization in cells. To further confirm whether the variant has an effect on the number of original lysosomes in cells, we respectively transferred wild-type (E)TMEM192(WT) and mutant (A)ΔTMEM192(127-267aa) into two groups of HEK293 cells, and set up two groups in which they were co-transfected with (D)mScarlat3-nanobody-ALFA-10His plasmid. After 48 hours, they were stained with the lysosomal dye LysoTracker-Green. The results showed that the number of lysosomes in cells overexpressing wild-type TMEM192 increased significantly, while the number of lysosomes in cells overexpressing mutant was close to that of the control group, indicating that overexpression of mutant (A)ΔTMEM192(127-267aa) does not significantly affect the number of lysosomes in the original cells and can be used to develop a lysosome visualization and extraction tool.

[0102] Lysosomes can be significantly enriched and extracted using the LysoEI system

[0103] like Figure 4As shown, the LysoEI system (A+D) plasmid was stably expressed in HEK293 cells at the same time. The results of cell expression (A) and (D) can be seen in the figure. At the same time, we extracted lysosomes through the lysosome extraction scheme designed by this invention. As can be seen in the figure, lysosomes can be significantly enriched compared with cytoplasmic components. The above results show that the LysoEI system designed by us can simultaneously achieve the enrichment and visualization of lysosomes of target cells. The lysosomes purified by this technology can be further combined with other multi-omics analysis, which plays an important supporting role in the in-depth exploration of the complex functions of organelles.

[0104] This study, for the first time, engineered a portion of the transmembrane region of TMEM192 as a lysosomal-specific targeting element, avoiding the perturbation of the cellular proteome caused by overexpression of a fully functional protein. Furthermore, we designed a nanobody-based fluorescent protein and enrichment tag to enable precise extraction and cytological imaging of lysosomes. This technology can be used to extract and image lysosomes in target organisms and can be combined with other omics techniques to enable systematic study of lysosomes, holding significant potential for application.

Claims

1. A method for lysosome visualization and enrichment extraction, characterized in that The steps include: (1) constructing a marker plasmid for overexpressing ΔTMEM192 protein; the ΔTMEM192 protein is a truncation of amino acids 19-267 of the wild-type TMEM192 protein; the marker plasmid also contains a sequence for expressing an ALFA tag; (2) constructing a recognition plasmid; the recognition plasmid contains an ALFA tag antibody gene sequence, a fluorescent protein sequence, and a His tag sequence; (3) Introducing the constructed marker plasmid and recognition plasmid into the target cells; culturing, dyeing, and visualizing the lysosomes under a confocal microscope; or, enriching and extracting the lysosomes through the affinity interaction between the His tag on the lysosome and the nickel beads.

2. The method according to claim 1, wherein: The sequence of the ΔTMEM192 protein is specifically the protein sequence shown in SEQ ID No.

1.

3. The method according to claim 1, wherein: The marker plasmid described in step (1) contains a sequence for expressing the protein shown in SEQ ID No. 2; the identification plasmid described in step (2) contains a sequence for expressing the protein shown in SEQ ID No.

5.

4. The method according to any one of claims 1 to 3, wherein: The fluorescent protein sequence is the red fluorescent protein mScarlet3.

5. The method according to any one of claims 1 to 4, characterized in that The target cells described in step (3) are HEK293 cells.

6. A preparation for lysosome visualization and enrichment extraction, characterized in that: Contain at least two of the following plasmids; (1) A marker plasmid for overexpressing ΔTMEM192 protein; the ΔTMEM192 protein is a truncation of amino acids 19-267 of the wild-type TMEM192 protein; the marker plasmid also contains a sequence for expressing an ALFA tag. (2) Identification plasmid; the identification plasmid contains ALFA tag antibody gene sequence, fluorescent protein sequence and His tag sequence.

7. The preparation of claim 6, wherein: The sequence of the ΔTMEM192 protein is specifically the protein sequence shown in SEQ ID No. 1; the fluorescent protein sequence is the red fluorescent protein mScarlet3.

8. The preparation of claim 6, wherein: The marker plasmid contains a sequence for expressing the protein shown in SEQ ID No. 2; the recognition plasmid contains a sequence for expressing the protein shown in SEQ ID No.

5.

9. Use of ΔTMEM192 protein in preparing a preparation for lysosome visualization and enrichment extraction; the sequence of the ΔTMEM192 protein is specifically the protein sequence shown in SEQ ID No. 1.

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