A lysosome-targeting polypeptide, lysosome fluorescent probe and kit

Abstract

The application discloses a kind of targeting lysosome polypeptide, lysosome fluorescent probe and kit, belong to the field of biotechnology.The targeting lysosome polypeptide sequentially includes A, B, C, D four parts from N-terminal to C-terminal, wherein, A part is cell penetrating peptide sequence, B part is lysosome targeting positioning sequence, C part is connecting peptide sequence, D part is fluorescent protein sequence.The polypeptide can quickly enter cell and target positioning in lysosome, has good biological safety and stability, can be applied as lysosome fluorescent probe, is suitable for the fluorescence imaging of lysosome in different living cells, and has specificity for lysosome staining.And lysosome polypeptide probe kit can be developed therefrom, to provide accurate lysosome organelle positioning and convenient application for scientific experimental research.

Description

[0001] This application is a divisional application of the invention patent application filed on August 17, 2023, with application number 2023110362101 and invention title "A polypeptide targeting lysosomes and its application". Technical Field

[0002] This invention relates to the field of biotechnology, specifically to a polypeptide targeting lysosomes and its applications, including a polypeptide lysosome fluorescent probe and kit. Background Technology

[0003] Lysosomes, as acidic organelles in eukaryotic cells (acidic environment, pH 4.5-6.5), participate in maintaining essential cellular life activities. The main functions of lysosomes are digestion and degradation. Lysosomes contain dozens of soluble hydrolases, including lipases, glycosidases, proteases, nucleases, and phosphatases, which break down various exogenous and endogenous macromolecules. Macromolecular degradation is essential for many cellular processes, such as clearing protein aggregates and dysfunctional organelles, recovering amino acids and energy required for biosynthesis, and clearing pathogens. In recent years, targeted protein degradation (TPD) technology has attracted considerable attention in the biomedical and pharmaceutical fields. Among different protein degradation strategies, lysosome-based targeted protein degradation strategies are considered to have great potential for disease treatment. This is mainly due to the broad degradation range of lysosomal protein pathways, covering proteins both inside and outside cells, as well as organelles. This has led to the development of a representative protein degradation strategy—lysosome-targeting chimera (LYTAC)—which can be used to degrade cell surface and extracellular proteins. LYTAC is a complex molecule composed of an oligosaccharide peptide group and an antibody that binds to a specific membrane or extracellular protein. The mechanism of action involves the oligosaccharide peptide group binding to the cell surface receptor CI-M6PR, while the antibody binds to the target protein. The complex formed by these two parts is engulfed by the cell membrane, forming a vesicle. Subsequently, with the assistance of the lysosome-targeting receptor CI-M6PR, it is ultimately carried to the lysosome to degrade the target protein. This has significant implications for the treatment of diseases related to membrane or extracellular proteins. Therefore, the development of novel lysosome-targeted protein degradation technologies has broad application prospects.

[0004] In addition to their protein degradation function, lysosomes are also widely involved in functions such as autophagy, innate and adaptive immunity, and amino acid sensing. Due to their important role in physiological processes, lysosomal dysfunction is involved in the development of many diseases, such as lysosomal storage diseases, neurodegenerative diseases, myocardial and skeletal muscle injuries, and immune diseases. Therefore, visualizing and monitoring the behavior of lysosomes and changes in their microenvironment is crucial for understanding lysosome-related diseases, which is conducive to promoting the study of their mechanisms and laying the foundation for the development of therapeutic drugs. Considering the importance of lysosomes, it is necessary to develop fluorescent probes that can be used for lysosomal imaging applications. However, currently available commercial lysosomal probes are small molecule chemical reagents, which, in addition to being difficult to synthesize and expensive, also have the following shortcomings: (1) Non-specificity: The commercially available lysosomal probes used in confocal experiments are acidic subcellular structure targets, rather than lysosomal targets. For example, in the case of live cells, when commercially available lysosomal probes are used for staining, if the concentration of the probe is too high or the staining time is too long, fluorescence will appear not only in the lysosomes but also in the cytoplasm. This is because small molecule probes can also cause non-specific staining of other acidic organelles (such as peroxisomes, mitochondria, and autophagosomes). Neutral Red and Acridine Orange can both fluoresce lysosomes, but both lack specificity. (2) Damage to lysosomes: The structural integrity and functional stability of organelles are the basis of cell life activities. Commercially available lysosomal probes can cause lysosomes to be alkalized, which increases the pH value of lysosomes. This may damage the intact structure of lysosomes and release the contents of lysosomes, thereby affecting the normal function of cells. (3) Fluorescence is easily quenched: Commercially available probe fluorescent dyes all have quenching problems. In addition, the instructions for commercially available lysosomal probes (Lyso-Tracker, catalog number: C1046 / 1047, Beyotime) all include the following precautions: lysosomal probes can only preferentially stain acidic lysosomes at extremely low concentrations (usually about 50 nM). When the concentration of the probe is too low or the staining time of the lysosomal probe is short, fluorescence cannot be seen during confocal observation. This may be because the commercially available lysosomal probes fail to enter the cells, or although a small amount of dye enters the lysosomes, its fluorescence is easily quenched under laser irradiation and therefore cannot be seen. (4) Unclear retention mechanism: The retention mechanism of commercially available small molecule probes is unclear. Current explanations suggest that this mechanism may be related to the protonation and retention of acidic organelles.

[0005] Therefore, in order to fully utilize the efficient protein degradation capabilities of lysosomes, it is desirable to develop a novel peptide drug for targeted degradation of membrane proteins and, based on this, a membrane protein targeted degradation kit, as well as a peptide probe that targets lysosomes. Summary of the Invention

[0006] The primary objective of this invention is to provide a lysosome-targeting polypeptide that exhibits good biocompatibility and stability in vivo, and demonstrates a significant degradation effect on cell surface membrane proteins. This allows it to be used in scientific research or in drug development to improve the therapeutic efficacy for diseases related to membrane protein abnormalities. Furthermore, based on the well-defined lysosomal localization characteristics of this polypeptide sequence, the substitutability of fluorescent proteins, and the ease of obtaining large quantities of the polypeptide sequence with good uniformity and stability, it is hoped that, leveraging these advantages, kits related to lysosomal polypeptide probes will be developed, including lysosomal fluorescent probe kits and protein degradation kits.

[0007] The fusion protein provided in this application comprises four parts, A, B, C, and D, from its N-terminus to its C-terminus. Part A is the cell-penetrating peptide sequence, part B is the lysosomal targeting sequence, part C is the linker sequence, and part D is the functional sequence. These parts can be modified according to different applications; for example, part D may be a fluorescent protein sequence when used as a lysosomal fluorescent probe, or a nanobody sequence when used as a protein degradation kit. By sequentially linking and fusing the sequences of parts A, B, C, and D, it can be adapted to various applications.

[0008] Part A of the sequence is a cell-penetrating peptide, such as Pep-1, whose amino acid sequence is: KETWWETWWTEWSQPKKKRKV (SEQ ID No: 1 in the sequence listing). Besides Pep-1, Part A can also be a cell-penetrating peptide, including but not limited to cationic, amphiphilic, and hydrophobic peptides, such as TAT, MAP, Transportan, MPG, Bip, Pep-7, and FGF. Table 1 lists the sequences of some commonly used cell-penetrating peptides (SEQ ID Nos: 1–10 in the sequence listing).

[0009] Table 1. Cell-penetrating peptide sequences

[0010] sequence name Classification KETWWETWWTEWSQPKKKRKV Pep-1 Amphibian GALFLGFLGAAGSTMGAWSQPKKKRKV MPG Amphibian MVKSKIGSWILVLFVAMWSDVGLCKKRPKP Bovine Prp (1-30) Amphibian WKCRRQAFRVLHHWN AFR Amphibian PPRLPRPRPRPLPFPRPG Bac7-24 Amphibian CSIPPEVKFNKPFVYLI C105Y Hydrophobic RLSGMNEVLSFRWL SG3 Hydrophobic RKKRRQRRR TAT(49-57) cationic R5, R7, R8, R9, R10, R11, R12 Polyargine cationic CRQIKIWFQNRRMKWKK Penetratin cationic TRQARRNRRRRWRERQR Rev(34-50) cationic

[0011] Part B is a lysosomal targeting sequence, such as saposin A, whose amino acid sequence is: GSLPCDICKDVVTAAGDMLKDNATEEEILVYLEKTCDWLPKPNMSASCKEIVDSYLPVILDI IKGEMSRPGEVCSALNLCES (SEQ ID No: 11 in the sequence listing); other lysosomal targeting sequences include, but are not limited to, saposin B, saposin C, saposin D, lamp1, lamp2, and lamp3.

[0012] The C-part linker peptide is preferably a flexible linker peptide (GGGGS). n Where n is an integer from 1 to 4. Flexible linker peptides play a crucial role in connecting AB and D, avoiding interference with the active site and binding ability of the D portion of the protein.

[0013] The D-part sequence is fused to the C-terminus of the entire protein polypeptide and is divided into two categories based on specific applications:

[0014] (1) When used as a lysosomal probe, part D is a fluorescent protein sequence, including but not limited to GFP, BFP, CFP, YFP, mCherry, etc. Table 2 lists the excitation wavelength and emission wavelength of some common fluorescent proteins.

[0015] Table 2. Emission and excitation wavelengths of fluorescent proteins

[0016] Fluorescent protein name Excitation wavelength (nm) Emission wavelength (nm) BFP 381 445 CFP 456 480 GFP 488 507 YFP 515 529 mHoneydew 504 562 mBanana 540 553 mOrange 548 562 tdTomato 554 581 mTangerine 568 585 mStrawberry 574 596 mCherry 587 610 mGrape1 595 620 mRaspberry 596 625 mGrape2 605 636 mPlum 590 648

[0017] (2) When used as a protein degradation kit, part D is a polypeptide sequence with the ability to recognize target proteins, such as the smCherry nanobody LaM2 sequence (SEQ ID No: 13 in the sequence listing), including but not limited to other different subtypes of mCherry nanobody, GFP nanobody and other nanobody sequences that can target membrane proteins, as well as short peptides, scfv (single chain antibody) and conventional antibodies with target recognition capabilities.

[0018] The lysosome-targeting polypeptide described in this invention can have the following effects:

[0019] 1) Rapidly enters any cell with the help of cell-penetrating peptides;

[0020] 2) Guided by lysosomal localization sequences, it targets and locates itself in lysosomal organelles after entering the cell;

[0021] 3) Depending on the application, it can be developed into a polypeptide fluorescence imaging probe for live cell lysosomal organelles and a membrane protein degradation kit based on a lysosomal degradation system.

[0022] The lysosomal polypeptide fluorescent probes prepared in this invention can exhibit a rich variety of colors, making them suitable for fluorescence imaging of lysosomes in different live cells, and they also possess specificity for lysosomal staining. This lysosomal polypeptide probe kit will overcome the shortcomings of commercially available lysosomal probes, greatly enriching the application market for lysosomal probes and providing accurate lysosomal organelle localization and convenient application for scientific experimental research. The polypeptide lysosomal probes and membrane protein degradation kit are applicable to a wide range of cell lines, including but not limited to human or mouse cell lines, such as: SF9, 293, A549, HeLa, B16-F10, MDA-MB-231, and HEK293T. Attached Figure Description

[0023] In the accompanying drawings of this invention, Pep1-Saposin A-(GGGS)2linker is uniformly referred to as PS, and will not be further emphasized in the following figure notes.

[0024] Figure 1 This is a schematic diagram of different components of the polypeptide sequence targeting lysosomes described in this invention.

[0025] Figure 2 The results of protein structure prediction using AlphaFold2 are shown for the PS-YFP fusion protein, where the D portion is a yellow fluorescent protein.

[0026] Figure 3 The results of protein structure prediction using AlphaFold2 are shown for the PS-GFP fusion protein whose D portion is green fluorescent protein.

[0027] Figure 4 The results of protein structure prediction using AlphaFold2 are shown for the PS-BFP fusion protein, where the D portion is blue fluorescent protein.

[0028] Figure 5 The results of protein structure prediction using AlphaFold2 are shown for the PS-CFP fusion protein whose D portion is cyan fluorescent protein.

[0029] Figure 6 PS-GFP fusion protein and a commercial lysosomal probe (Lyso-Tracker red) were incubated in 293 cells with and without serum at 37°C for 2 h. The colocalization of the two different fluorescence was then observed using a laser confocal microscope.

[0030] Figure 7 The results are for cytotoxicity assays of the PS-GFP peptide in different cell lines (incubated at 37°C for 6 hours).

[0031] Figure 8The results of the in vitro safety evaluation of different concentrations of PS-GFP fusion protein using a hemolysis assay are presented.

[0032] Figure 9 The results show the statistical results of hemolysis rates in groups treated with different concentrations of PS-GFP fusion protein in the hemolysis experiment.

[0033] Figure 10 To investigate the cell entry pathway of PS-GFP peptide using different cell entry inhibitors (pretreatment of 293 cells for 1 h) and to explore whether energy-dependent cell entry (pretreatment of 293 cells at 4°C for 1 h) occurred.

[0034] Figure 11 The PS-GFP fusion protein was incubated in SF9 and 293 cells at 37°C for 4 h, and the co-localization results of PS-GFP and commercially available lysosomal probes were observed using laser confocal microscopy.

[0035] Figure 12 The results of protein structure prediction using AlphaFold2 for the PS-mCherry nanobody fusion protein.

[0036] Figure 13 The degradation of mCherry protein was observed at different time points in cell lines where the PS-mCherry nanobody fusion protein was localized to the mCherry membrane.

[0037] Figure 14 AlphaFold2 was used to predict the structure of the PS-KN035(PDL1 nanobody) fusion protein. Detailed Implementation

[0038] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the 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 field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0039] Example 1: Purification of Pep1-Saposin A-GFP (PS-GFP) protein

[0040] In this embodiment, Pep1-Saposin A-(GGGS)2linkers are abbreviated as PS. This will not be emphasized again in subsequent embodiments. (1) Plasmid construction: The gene sequence encoding PS-GFP was constructed into the pet28a plasmid vector, and bacterial strains were obtained by transformation using BL21(DE3) competent cells;

[0041] (2) Induction of expression: PS-GFP bacteria were added to 5 mL of LB medium and activated overnight, then 1 L of LB medium was added to expand bacterial culture; when the OD of the bacterial culture reached a certain level, the expression was induced. 600 When the concentration was 0.8, the protein expression induction reagent isopropyl-β-D-thiogalactoside IPTG (final concentration 0.01mM) was added, the shaker temperature was set to 20℃, and the protein expression induction time was 16h.

[0042] (3) Harvesting bacterial cells: Collect bacteria by centrifugation at 4000g for 10 min, resuspend in 50mL TBS, and add protease inhibitor PMSF to the bacterial solution;

[0043] (4) Ultrasonic lysis of bacteria: 10 minutes, power 150W;

[0044] (5) Centrifugation: Centrifuge at 13000 rpm for 30 min at low temperature;

[0045] (6) Binding: Add the protein supernatant after centrifugation to the protein purification gravity column and bind at 4°C for 1 hour;

[0046] (7) Protein collection: First, let the supernatant flow to just cover the Ni column, and start eluting with 50 mL of low-salt solution, which is denoted as NTA0; prepare 20, 40, and 250 mM imidazole low-salt solutions for elution, which are denoted as NTA20, NTA40, and NTA250, respectively; among them, NTA250 elution contains the target protein;

[0047] (8) Concentration: The NTA250 eluent was centrifuged at 3500g and 4℃ using a protein concentration tube (10kD) and concentrated to 0.5mL. The protein was then further purified and collected using a molecular sieve instrument.

[0048] (9) Storage: Aliquot the protein into centrifuge tubes. For short-term storage, the protein can be stored in a 4°C freezer. For long-term storage, liquid nitrogen flash freezing is required and the protein should be stored in a -80°C freezer.

[0049] Example 2: Protein structure prediction of PS-GFP, PS-BFP, PS-CFP, and PS-YFP fusion proteins using AlphaFold2.

[0050] In order to design fusion proteins in a reasonable manner to prevent site interference from affecting the cell entry efficiency of fusion proteins, we imported the above-mentioned protein sequences to be predicted into the AlphaFold2 protein prediction software in FASTA format for protein structure prediction.

[0051] The fusion protein sequence composition targeting the lysosome is as follows: Figure 1As shown, these include cell-penetrating peptides, lysosomal localization sequences, flexible linker sequences, and, depending on the application, fluorescent protein sequences or nanobodies capable of recognizing targets. The experimental results for AlphaFold2 are as follows... Figures 2 to 5 As shown, all of the above protein sequences have complete structural models. Moreover, the fusion of the penetrating peptide Pep-1, the lysosomal localization sequence saposin A, and different fluorescent proteins (GFP, BFP, CFP, and YFP) does not affect the binding sites of the fluorescent proteins. It can be considered that the fusion of these fluorescent proteins can be reasonably designed in this way, so the purification and collection of the above fusion protein sequences can continue.

[0052] Example 3: Co-localization of PS-GFP fusion protein with commercially available lysosomal probe Lyso-Tracker red

[0053] To verify the uptake of the fusion peptide in cells, we used PS-GFP fusion protein (fused GFP green fluorescent protein) as an example. PS-GFP fusion protein (concentration: 100 μg / mL) was added to 293 cells, and different incubation conditions were set: a control group containing 10% serum and a control group without serum. After incubation at 37°C for 2 h, the cells were incubated for 10 min with commercially available lysosomal probe Lyso-Tracker red (red fluorescence, catalog number C1046 / 1047, Beyotime; concentration 10 μmol / L). After washing three times with phosphate-buffered saline (PBS), observation and imaging were performed using a laser confocal microscope.

[0054] Laser confocal results are as follows Figure 6 The results showed that the PS-GFP fusion protein can enter the cytoplasm rapidly and efficiently. In the presence or absence of serum, PS-GFP can enter the cytoplasm and achieve effective co-localization with commercially available lysosomal probes.

[0055] Example 4: Cytotoxicity assay of PS-GFP fusion protein in different cell lines

[0056] To evaluate the safety of fusion proteins, we used PS-GFP protein (fusion GFP green fluorescent protein) as an example and added different concentrations of PS-GFP protein (10, 25, 50, and 100 μg / mL) to different cell lines A549 (lung cancer cell line), B16 (melanoma cell line), and HeLa (cervical cancer cell line) for cytotoxicity experiments. First, cells were seeded in 96-well plates (cell density: 10,000 cells / well). After incubating at 37°C for 24 h, different concentrations of PS-GFP protein were added (PS-GFP protein was centrifuged at 12,000 rpm for 5 min before addition to remove impurities and bacteria). After incubating at 37°C for 6 h, the cytotoxicity assay was performed according to the CCK-8 cytotoxicity assay kit at a wavelength of 450 nm.

[0057] Cytotoxicity test results as follows Figure 7 The results showed that all three cell types exhibited good growth at different drug concentrations, with a survival rate close to 100%, indicating that the PS-GFP protein has good biocompatibility.

[0058] Example 5: Evaluation of the biosafety of PS-GFP fusion protein using a hemolysis assay

[0059] Take a 15 mL centrifuge tube, add 1 mL of heparin-containing anticoagulant blood, add 9 mL of phosphate buffered saline (PBS, pH 7.4), mix well, and centrifuge at 1500 rpm for 5 min. Remove the supernatant, add PBS again along the tube wall, and repeat the above operation three times. Take 1 mL of red blood cells from the bottom and add 9 mL of PBS to prepare a 10% red blood cell solution. The 10% red blood cell solution was used for sample preparation, with positive control, negative control, and different concentrations of PS-GFP treatment groups (concentrations: 10, 25, 50, 100, and 200 μg / mL). Aliquot the above solutions into EP tubes, incubate at 37℃ for 1 h, and centrifuge (3500 rpm for 5 min). The hemolysis status of the solutions in the EP tubes was then photographed and recorded. Subsequently, 0.1 mL of each sample was added to a 96-well plate, and the absorbance of the samples was detected using a microplate reader, and the hemolysis rate was statistically analyzed (detection wavelength: 540 nm). Negative control (NC): phosphate buffer solution; Positive control (PC): distilled water. The hemolysis rate was calculated using the formula below, and the biosafety of the PS-GFP protein was determined according to the International Organization for Standardization (ISO) standard (standard: hemolysis rate <5%).

[0060]

[0061] like Figure 8 and Figure 9As shown, different concentrations of PS-GFP exhibited minimal erythrocyte rupture or agglutination. The PC group showed complete hemolysis, while the hemolysis rates corresponding to different concentrations of PS-GFP (10, 25, 50, 100, and 200 μg / mL) were 0.73%, 0.86%, 0.86%, 1.33%, and 1.79%, respectively. The hemolysis rates of all PS-GFP protein concentrations were below 5%, indicating that PS-GFP has good biocompatibility with cells.

[0062] Example 6: Detection of the cell entry pathway of PS-GFP fusion protein using different cell entry inhibitors

[0063] To investigate the pathway by which fusion proteins enter cells, we used PS-GFP protein as an example. Cells were pretreated with different cellular pathway inhibitors (genistein, nystatin, and amiloride) for 1 hour, followed by the addition of PS-GFP protein (concentration: 100 μg / mL). After incubation at 37°C for 2 hours, the cells were incubated with the commercially available lysosomal probe Lyso-Trackerred (concentration: 10 μmol / L) for 10 minutes. After washing three times with phosphate-buffered saline (PBS), the cells were observed and photographed using laser confocal microscopy. Furthermore, to investigate whether PS-GFP protein enters cells in an energy-dependent manner, we pretreated the cells with low temperature, and subsequent procedures were the same as described above.

[0064] Laser confocal results are as follows Figure 10 The results showed that punctate green fluorescence appeared in all the above-mentioned treatment groups, and good co-localization with commercially available lysosomal probes was observed. Furthermore, PS-GFP could still effectively enter cells in the low-temperature treatment group and effectively co-localize with commercially available lysosomal probes. This indicates that PS-GFP is not limited by conventional cell entry pathway inhibitors and intracellular energy, and belongs to the non-endocytic pathway for cell entry.

[0065] Example 7: Colocalization of PS-GFP fusion protein with commercially available lysosomal probes in SF9 and 293 cells

[0066] To evaluate the feasibility of lysosomal peptide fluorescent probes in different cell lines, we used PS-GFP protein as an example to verify its effectiveness in two cell lines (SF9 and 293 cells). PS-GFP (concentration: 100 μg / mL) was added to the cells, and after incubation at 37°C for 4 h, the cells were incubated with commercially available lysosomal probe Lyso-Tracker red (concentration: 10 μmol / L) for 10 min. After washing three times with phosphate-buffered saline (PBS), laser confocal microscopy was performed for observation and imaging.

[0067] Laser confocal results are as follows Figure 11The results showed that the PS-GFP protein was distributed in a punctate pattern in the cytoplasm and exhibited effective co-localization with commercially available lysosomal probes. After prolonged incubation, the PS-GFP protein did not leak into the cytoplasm and remained specifically localized to lysosomal organelles, demonstrating its excellent lysosomal localization performance and fluorescence stability.

[0068] Example 8: Protein structure prediction of PS-mCherry nanobody and PS-KN035 (PDL1 nanobody) proteins using AlphaFold2

[0069] We developed a novel membrane protein degradation technology based on nanobodies, which primarily utilizes the lysosomal system to degrade cell surface membrane proteins. Using mCherry protein as a model target, we prepared a PS-mCherry nanobody fusion protein (SEQ ID No: 14 in the sequence listing) for degrading membrane-localized mCherry fluorescent protein. Rational protein structure design is crucial for subsequent protein function and purification. Before validating the efficacy, we first used Alphafold2 to predict the protein sequences of the PS-mCherry nanobody and PS-KN035 (SEQ ID No: 15 in the sequence listing) to rationally design this membrane protein degradation technology.

[0070] Based on the Alphafold2 protein sequence prediction results, as follows: Figure 12 and Figure 14 The results showed that PS-mCherry nanobody and PS-KN035 exhibited complete protein structures, with the penetrating peptide, lysosomal localization sequence and nanobody sequence not interfering with each other. The α-helix structure exhibited by the different fusion proteins facilitated rapid entry into cells.

[0071] Example 9: Degradation of PS-mCherry nanobody (LaM2 nanobody) protein peptides at different time points in mCherry-PDL1 membrane-localized cell lines.

[0072] To investigate and verify that lysosome-targeting peptide sequences can target and degrade membrane proteins, we prepared and purified a PS-mCherry nanobody fusion protein for degrading membrane-localized mCherry fluorescent protein. The degradation effect was mainly observed by monitoring the fluorescence changes of mCherry protein at different time points. In the transiently transfected cell line 293 containing membrane-localized mCherry protein, we divided the cells into a control group and a treatment group (three replicates per group). The treatment group was treated with PS-mCherry nanobody at a concentration of 100 μg / mL, and the fluorescence intensity of mCherry was recorded every hour for 24 hours using a long-term live-cell fluorescence quantitative PCR instrument.

[0073] Experimental results are as follows Figure 13 The results showed that, compared with the control group (untreated), the fluorescence values ​​collected at different time points in the PS-mCherry nanobody treatment group were significantly lower than those in the control group. Therefore, PS-mCherry nanobody has a significant degradation effect on mCherry proteins on the cell membrane surface.

Claims

1. A polypeptide, characterized in that, The sequence consists of four parts, A, B, C, and D, from the N-terminus to the C-terminus. Part A is a cell-penetrating peptide sequence, part B is a lysosomal targeting sequence, part C is a linker peptide sequence, and part D is a fluorescent protein sequence. Part A is Pep-1, and its amino acid sequence is shown in SEQ ID No:

1. Part B is saposin A, and its amino acid sequence is shown in SEQ ID No:

11. Part D is GFP, BFP, CFP, YFP, or mCherry.

2. The polypeptide according to claim 1, characterized in that, Part C is a flexible linker peptide (GGGGS). n , where n is an integer from 1 to 4.

3. A lysosomal fluorescent probe, characterized in that, Includes the polypeptide described in claim 1 or 2.

4. A reagent kit, characterized in that, The kit is a lysosomal fluorescent probe kit, including the lysosomal fluorescent probe as described in claim 3.

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