Medicine for improving expression of lysosome-associated transmembrane protein 4B and screening method thereof
By inserting a fluorescent marker into the LAPTM4B gene locus and binding it to the CD63 signal, a high-throughput screening system was used to screen for drugs such as Ruboxistaurin. This solved the problem of the lack of regulatory molecules for LAPTM4B, achieved precise regulation of LAPTM4B expression, and improved the efficacy and safety of tumor treatment.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ANHUI MEDICAL UNIV
- Filing Date
- 2025-11-10
- Publication Date
- 2026-05-12
AI Technical Summary
The lack of a regulatory molecular screening platform for lysosome-associated transmembrane protein 4B (LAPTM4B) in existing technologies makes it impossible to effectively regulate its expression and function, affecting the precision and safety of tumor treatment.
Fluorescent markers were precisely inserted into the LAPTM4B gene locus, and the membrane protein CD63 was used as an internal reference signal. A high-throughput automated drug screening system was used to screen for drugs that could enhance LAPTM4B expression, including Ruboxistaurin, Motesanib, SGC0946, GSK-461364, Imatinib, and Gefitinib.
This study enabled precise spatiotemporal manipulation of LAPTM4B expression, allowing for the screening of highly specific drugs, improving therapeutic efficacy in tumor cell models, reducing side effects, and providing new directions for therapeutic target research.
Smart Images
Figure CN122005552A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of drug screening technology, and in particular to a drug expressing lysosome-associated transmembrane protein 4B and a screening method thereof. Background Technology
[0002] Currently, global cancer drug development is rapidly shifting from the traditional "cytotoxic chemotherapy" model to a precision treatment phase centered on specific molecular targets. Traditional chemotherapy drugs primarily kill tumor cells by inhibiting cell division in a non-specific manner. However, these drugs are often accompanied by severe toxic side effects and drug resistance. In recent years, with the rapid development of genomics, structural biology, and signal transduction research, it has become increasingly clear that the occurrence and progression of tumors are a comprehensive result of the abnormal activation of multiple driver genes and key signaling pathways.
[0003] Therefore, target-based drug screening has gradually replaced empirical screening and become the dominant direction in modern drug discovery. This strategy achieves precise regulation of tumor cells by identifying and intervening in key molecular targets, significantly improving drug efficacy and safety. Among many potential molecular targets, lysosomal-associated transmembrane protein 4B (LAPTM4B), as a novel oncogene, has received widespread attention in recent years. This protein was first identified as a significantly overexpressed molecule in hepatocellular carcinoma (HCC), and subsequent studies have found that it is abnormally upregulated in breast cancer, lung cancer, gastric cancer, ovarian cancer, and acute myeloid leukemia. LAPTM4B promotes tumor cell growth, migration, invasion, and drug resistance by regulating autophagy, mTORC1 signaling activation, drug efflux, and membrane protein transport. Its high expression level is significantly associated with poor prognosis in patients with various cancers.
[0004] However, to date, no regulatory molecules targeting LAPTM4B have been reported, and the lack of an efficient and systematic screening platform has become a significant bottleneck in this field. Given the crucial role of LAPTM4B in tumorigenesis and drug resistance, it is essential to establish a small molecule screening system capable of dynamically monitoring its expression, localization, and regulation at the cellular level, thereby identifying regulatory molecules targeting LAPTM4B. Summary of the Invention
[0005] Therefore, the technical problem to be solved by the present invention is to overcome the lack of a regulatory molecule for LAPTM4B in the prior art.
[0006] To address the aforementioned technical problems, this invention provides a lysosome-associated transmembrane protein 4B agonist and its screening method. This invention precisely inserts a fluorescent marker into the LAPTM4B gene locus to achieve real-time monitoring of the dynamic changes of endogenous proteins. Simultaneously, it introduces the membrane protein CD63 as an internal reference signal. Using a high-throughput automated drug screening and image analysis system, compounds in a large-scale drug library are quantitatively screened, ultimately identifying drugs that can enhance LAPTM4B expression. Using the screening method of this invention, drugs that can enhance lysosome-associated transmembrane protein 4B have been identified, providing a new direction for subsequent research on therapeutic targets related to lysosome-associated transmembrane protein 4B. Furthermore, the drugs screened by this invention exhibit good specificity, enhancing LAPTM4B expression without affecting CD63, thus enabling precise spatiotemporal manipulation of LAPTM4B activity, which is of great significance in constructing in vitro / in vivo transient activation models.
[0007] The first objective of this invention is to provide a drug that increases the expression of lysosome-associated transmembrane protein 4B (LAPTM4B), said drug being selected from one or more of Ruboxistaurin, Motesanib, SGC0946, GSK-461364, Imatinib, and Gefitinib.
[0008] Furthermore, Ruboxistaurin (CAS No. 169939-94-0) is an orally active, selective PKC beta inhibitor.
[0009] Motesanib (CAS No. 453562-69-1) is a potent ATP-competitive inhibitor of VEGFR1 / 2 / 3.
[0010] SGC0946 (CAS No. 1561178-17-3) is a selective DOT1L inhibitor.
[0011] GSK-461364 (CAS No. 929095-18-1) is a selective, reversible, ATP-competitive PLK1 inhibitor.
[0012] Imatinib (CAS No. 152459-95-5) is a tyrosine kinase inhibitor that selectively inhibits the activity of BCR / ABL, v-Abl, PDGFR, and c-kit kinases.
[0013] Gefitinib (CAS No. 184475-35-2) is an EGFR tyrosine kinase inhibitor.
[0014] A second objective of this invention is to provide the use of Ruboxistaurin, Motesanib, SGC0946, GSK-461364, Imatinib, or Gefitinib in the preparation of products that enhance the expression of lysosome-associated transmembrane protein 4B.
[0015] A third objective of this invention is to provide a drug that enhances the expression of lysosome-associated transmembrane protein 4B for use in the preparation of cancer models, said drug being selected from one or more of Ruboxistaurin, Motesanib, SGC0946, GSK-461364, Imatinib, and Gefitinib.
[0016] Furthermore, the tumor includes hepatocellular carcinoma, breast cancer, lung cancer, stomach cancer, or ovarian cancer.
[0017] A fourth objective of this invention is to provide the use of a drug that enhances the expression of lysosome-associated transmembrane protein 4B in the preparation of lysosome-associated transmembrane protein 4B modulators or vaccine adjuvants.
[0018] A fifth objective of this invention is to provide a reagent that promotes the proliferation and survival of tumor cells, the reagent being selected from one or more of Ruboxistaurin, Motesanib, SGC0946, GSK-461364, Imatinib, and Gefitinib.
[0019] A sixth objective of this invention is to provide a method for screening drugs that increase drug resistance in tumor cells. The method involves administering a drug that enhances the expression of lysosome-associated transmembrane protein 4B to tumor cells, followed by the addition of the drug to be screened. The drug that enhances the expression of lysosome-associated transmembrane protein 4B is selected from one or more of Ruboxistaurin, Motesanib, SGC0946, GSK-461364, Imatinib, and Gefitinib.
[0020] A seventh objective of this invention is to provide a method for screening drugs that enhance the expression of lysosome-associated transmembrane protein 4B, comprising the following steps:
[0021] S1. Construct recombinant cells containing lysosome-associated transmembrane protein 4B and lysosome-associated membrane protein 3, wherein the lysosome-associated transmembrane protein 4B is labeled with a first fluorescent protein, the lysosome-associated membrane protein 3 is labeled with a second fluorescent protein, and the first fluorescent protein and the second fluorescent protein are different.
[0022] S2. Co-incubate the drug to be screened with recombinant cells and detect the fluorescence signal. Calculate the ratio of the fluorescence signal of the first fluorescent protein to the fluorescence signal of the second fluorescent protein in the recombinant cells to screen for drugs that enhance the expression of lysosome-associated transmembrane protein 4B.
[0023] Further, in step S1, the first fluorescent protein-labeled lysosome-associated transmembrane protein 4B is obtained by inserting the first fluorescent protein between position 847 and position 848 of the lysosome-associated transmembrane protein 4B;
[0024] The second fluorescent protein-labeled lysosome-associated membrane protein 3 is obtained by inserting the second fluorescent protein between positions 786 and 787 of the lysosome-associated membrane protein 3.
[0025] Further, in step S1, the recombinant cell is achieved by introducing the Cas9 protein-coding gene, sgRNA, and donor DNA into the recipient cell, wherein the gene sequence of the sgRNA is shown in SEQ ID NO.1-4, the gene sequence of the donor DNA is shown in SEQ ID NO.5-6, and the recipient cell expresses lysosome-associated transmembrane protein 4B and lysosome-associated membrane protein 3.
[0026] Furthermore, the recipient cells include skin cancer cells A431.
[0027] Furthermore, the NCBI number of the lysosome-associated transmembrane protein 4B is 55353, and the NCBI number of CD63 is 967.
[0028] Furthermore, the first fluorescent protein and the second fluorescent protein are independently selected from any one of superfolded green fluorescent protein, green fluorescent protein, yellow fluorescent protein, blue fluorescent protein, and red fluorescent protein.
[0029] Furthermore, the red fluorescent protein includes mCherry red fluorescent protein.
[0030] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:
[0031] This invention precisely inserts a fluorescent marker at the LAPTM4B gene locus to achieve real-time monitoring of the dynamic changes of endogenous proteins. Simultaneously, it introduces the membrane protein CD63 as an internal reference signal. Using a high-throughput automated drug screening and image analysis system, compounds in a large-scale drug library are quantitatively screened, ultimately identifying drugs that can enhance LAPTM4B expression. Using this screening method, this invention identifies drugs that can enhance lysosome-associated transmembrane protein 4B, providing a new direction for subsequent research on therapeutic targets related to lysosome-associated transmembrane protein 4B. Furthermore, the drugs screened in this invention exhibit good specificity, enhancing LAPTM4B expression without affecting CD63, thus enabling precise spatiotemporal manipulation of LAPTM4B activity, which is of great significance in constructing in vitro / in vivo transient activation models. Attached Figure Description
[0032] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.
[0033] Figure 1 This is a diagram illustrating the construction principle and results of the dual-fluorescence reporter system of the present invention;
[0034] Figure 2 This is a graph showing the drugs screened by the dual fluorescence reporter system of this invention and their validation results. Detailed Implementation
[0035] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.
[0036] Example 1: Construction and validation of CRISPR-Cas9n-mediated LAPTM4B dual-fluorescent-labeled reporter cells
[0037] (1) Experimental objective
[0038] To achieve real-time visualization and monitoring of the endogenous expression and drug response of the target protein LAPTM4B, this embodiment constructed a LAPTM4B-sfGFP / CD63-mCherry dual-fluorescent reporter cell line, which can accurately reflect the expression and subcellular localization of LAPTM4B without altering the gene regulatory background. Using a dual-notch CRISPR-Cas9n (Cas9nickase) system, the LAPTM4B gene was precisely edited in the skin cancer cell line (A431), inserting a superfolded green fluorescent protein (sfGFP) sequence at its C-terminus. Homologous recombination enabled endogenous expression labeling without exogenous promoter interference. Simultaneously, a CD63-mCherry fluorescent labeling system was constructed in control cells as an internal control for membrane protein localization and signal normalization. After resistance selection and single-clone amplification, a stable integrated dual-reporter cell line (LAPTM4B-sfGFP / CD63-mCherry) was obtained. The correctness of the insertion and the physiological consistency of protein localization were verified by PCR, sequencing, Western blot, and confocal microscopy.
[0039] (2) Experimental materials and main instruments
[0040] Cell line: Skin cancer cell line (A431);
[0041] Gene editing system: CRISPR-Cas9n dual-cut system (reduces off-target risk);
[0042] Vector and donor construction: pSpCas9n(BB)-2A-Puro (PX462), sgRNA (Synthego) synthesized; donor DNA.
[0043] Main culture reagent: RPMI-1640 + 10% FBS;
[0044] Main instruments:
[0045] Leica TCS SP8 laser confocal microscope; PerkinElmer Opera Phenix high-content imaging system; Bio-Rad Mini-PROTEAN electrophoresis system; ChemiDoc MP imaging system; ABI 7500 Fast real-time PCR system.
[0046] (3) Experimental methods
[0047] ① sgRNA and donor template design
[0048] Double sgRNAs were designed targeting the terminal exon regions of the LAPTM4B and CD63 genes. Double-strand breaks were generated using the Cas9n system, prompting donor DNA containing the sfGFP (SEQ ID NO.5) or mCherry (SEQ ID NO.6) sequences to insert into the target sites via homologous recombination (HDR). The donor DNA design included left and right arms, a fluorescent gene, and a loxP-Puro-loxP selection marker sequence. Specific sequences are detailed in Table 1.
[0049] Table 1
[0050]
[0051]
[0052] ② Transfection and screening
[0053] pSpCas9n(BB)-2A-Puro (PX462), sgRNA and donor DNA were simultaneously transfected into cells. Cells were screened for puromycin resistance for 24-48 hours, and then expanded into single-clone culture in 96-well plates. The insertion accuracy was verified by Sanger sequencing of cells from each well.
[0054] ③ Expression verification and localization analysis
[0055] Immunoblotting and confocal imaging confirmed the expression and localization of the fluorescent fusion protein, revealing that LAPTM4B-sfGFP was mainly located in endosomes / lysosomes and highly co-localized with CD63-mCherry signals. Figure 1 B).
[0056] ④ Specificity verification (siRNA interference)
[0057] The changes in corresponding fluorescence signals were observed by specifically silencing LAPTM4B or CD63 using siRNA. Cells were plated one day before transfection at a density of 50-60%. For each well of a 6-well plate, 1.0 mL of complete culture medium containing serum and antibiotics was added 30-60 minutes before transfection. 2 μL of LAPTM4B / CD63 siRNA (20 μM) was added to the transfection working solution, followed by 3 μL of transfection reagent. After mixing thoroughly, the mixture was incubated at room temperature for 15 min to allow the transfection complex to form. Finally, the transfection complex was added dropwise to the cells. Five hours after transfection, the transfection medium was replaced with cell growth medium. The results showed a significant decrease in the individual signals, while the signals of the other remained stable, indicating that the system has high specificity. Figure 1 C–D).
[0058] Table 2 siRNA
[0059]
[0060] (4) Description of the detection method
[0061] Western blot: Proteins were extracted using RIPA lysis buffer and detected using anti-LAPTM4B, anti-GFP, anti-CD63, and anti-mCherry antibodies, with β-actin as an internal control.
[0062] Imaging analysis: 488 nm (GFP) and 561 nm (mCherry) excitation, colocalization coefficients were calculated using Fiji-Coloc2.
[0063] siRNA transfection: 50 nM, Lipofectamine RNAiMAX, detected after 48 h.
[0064] (5) Experimental Results and Conclusions
[0065] The dual-fluorescence reporter system can stably express and accurately reflect the endogenous level and localization characteristics of LAPTM4B, and can be used for screening high-content drugs. siRNA validation confirms its specificity and reliability. Figure 1 ).
[0066] Figure 1 A is a schematic diagram illustrating the endogenous fluorescent labeling of LAPTM4B and CD63 in the cell genome using the CRISPR-Cas9 system. The fluorescent tags sfGFP or mCherry are knocked into the space between the last exon of the LAPTM4B or CD63 gene and its 3' untranslated region (3'UTR), respectively, resulting in the dual-fluorescent reporter system of this embodiment.
[0067] Figure 1 B is a confocal microscopy image showing the intracellular localization of LAPTM4B-sfGFP and the control protein CD63-mCherry in endogenously labeled cells. The results indicate that the two proteins are mainly distributed in the endosome / lysosome region.
[0068] Figure 1 C is an electrophoresis diagram obtained by transfecting endogenous dual-labeled cell lines (LAPTM4B+CD63 tag) and wild-type cell lines (WT) with LAPTM4B or CD63 siRNA, respectively, and then detecting protein levels by Western blot using LAPTM4B antibody or GFP antibody, respectively. Black arrows represent endogenous LAPTM4B bands, and green arrows represent sfGFP-labeled LAPTM4B bands. The results indicate that the dual-fluorescent reporter system of this invention has high specificity.
[0069] Figure 1D involves transfecting endogenous double-labeled cell lines (L+C tag) and wild-type cell lines (WT) with LAPTM4B or CD63 siRNA, respectively, and then detecting protein levels by Western blot. Electrophoresis images were obtained using CD63 antibody or mCherry antibody, respectively.
[0070] Figure 1 E is a two-dimensional distribution map of the dual-channel fluorescence signals obtained by transfecting endogenous double-labeled cell lines (LAPTM4B+CD63 tag) with siRNA of LAPTM4B (L4B) or CD63, respectively, and capturing intracellular GFP and Cherry signals using high-content fluorescence microscopy.
[0071] Figure 1 F is a two-dimensional distribution map of the fluorescence signal in the plasma membrane obtained by transfecting endogenous double-labeled cell lines (LAPTM4B+CD63 tag) with siRNA of LAPTM4B or CD63, respectively, and capturing the GFP and Cherry signals of the plasma membrane (PM) using high-content fluorescence microscopy.
[0072] Figure 1 G is a graph showing the fluorescence signal and the bar graph of total fluorescence signal in a single channel (GFP or Cherry) obtained by transfecting endogenous double-labeled cell lines (LAPTM4B+CD63 tag) with siRNA of LAPTM4B or CD63, respectively, and capturing GFP and Cherry signals in the cytoplasmic membrane using high-content fluorescence microscopy.
[0073] Example 2: High-throughput drug screening and validation based on dual fluorescently labeled cell lines
[0074] (1) Experimental objective
[0075] We screened compounds that could specifically upregulate LAPTM4B protein levels to identify potential LAPTM4B activators.
[0076] (2) Experimental materials and instruments
[0077] Reporter cells: LAPTM4B-sfGFP / CD63-mCherry double-labeled cells;
[0078] Drug library: 527 FDA-approved or clinically advanced anti-tumor drugs (FIMM Oncology Collection).
[0079] Main instruments: Beckman Coulter Biomek FXP high-throughput pipetting robot; Echo 550 acoustic dispensing system; PerkinElmer Opera Phenix high-content microscopy system; Data analysis software: Harmony 4.8, CellProfiler, Python (Pandas / Numpy / Matplotlib).
[0080] (3) Experimental steps
[0081] ① Automated plating and drug treatment: 384-well plate format, approximately 1000 cells per well, with 5 concentration gradients (0.01-10 μM) and 3 replicates.
[0082] ②Fixed imaging 48 hours after processing.
[0083] ③ Automatic image acquisition and signal extraction, and calculation of the sfGFP / mCherry ratio in single cells.
[0084] ④ Statistical analysis was used to screen out compounds that significantly regulated LAPTM4B signaling without affecting CD63.
[0085] ⑤ Verify the specificity of drug action using Western blot.
[0086] (4) Experimental results
[0087] High-throughput screening results showed that Ruboxistaurin, Motesanib, SGC0946, GSK-461364, Imatinib, and Gefitinib significantly upregulated LAPTM4B levels, while CD63 signal was unaffected. Western blot validation results were consistent. Figure 2 As shown.
[0088] Figure 2 A is a schematic diagram of the drug screening experiment in this study. Cells were seeded into 384-well plates using an automated robotic system and processed from a tumor drug library containing 527 drug compounds, including FDA-approved drugs and novel drug candidates in clinical or experimental stages. After processing, the cells were fixed and imaged using a PerkinElmer Opera Phenix high-content microscopy system. Subsequently, automated image analysis algorithms were used to quantitatively analyze fluorescence signals to screen for compounds that could selectively upregulate LAPTM4B expression without affecting CD63 levels.
[0089] Figure 2B shows the scatter plot analysis results of the LAPTM4B-sfGFP and CD63-mCherry signal intensities after drug treatment. Blue dots represent compounds that reduce LAPTM4B expression levels, and red dots represent compounds that increase LAPTM4B levels.
[0090] Figure 2 C represents the top six candidate compounds that can significantly upregulate LAPTM4B expression without significantly affecting CD63.
[0091] Figure 2 D represents the expression levels of LAPTM4B and CD63 proteins in cells treated with selected representative drugs, which were then analyzed by Western blot to verify the reliability of the high-content screening results.
[0092] (5) Description of testing methods
[0093] Nine fields of view were imaged per well using a 40× objective lens. The Otsu algorithm was used for cell segmentation and signal normalization. Z-score analysis was used to screen for matching compounds. Protein detection was performed using the same method as in Example 1.
[0094] (6) Results Analysis
[0095] The upregulation rate of LAPTM4B was approximately 3.29%, suggesting that these drugs may positively regulate LAPTM4B expression. This system exhibited high signal stability, a low false positive rate, and results consistent with physiological correlations.
[0096] Comparative Example
[0097] (1) Purpose
[0098] Verify the technical advantages of the screening system of this invention in terms of specificity, sensitivity and data stability.
[0099] (2) Proportional settings
[0100] 1. Targeted control system: Drug screening targeting CD63 was re-screened using mCherry as a signal. The results showed that the CD63 upregulation hit rate was approximately 5.5%. In addition, the overlap rate between drugs upregulating CD63 and drugs upregulating LAPTM4B was only 0.94%, indicating that the results of this screening were drugs that specifically upregulate LAPTM4B.
[0101] 2. Fluorescence signal control system: Using a traditional overexpression system, such as pHAGE-Puro (Addgene, plasmid #118692) to digest the LAPTM4B transcript coding sequence (NM_018407.6) at the BamH1 site, the signal noise was significantly higher than that of the system of this invention, and some drugs caused non-specific GFP signal changes. Furthermore, traditional overexpression systems can only screen for drugs that affect protein stability, and cannot accurately capture drugs that endogenously regulate LAPTM4B (e.g., at the transcriptional level, mRNA stability level, etc.). This invention, however, can screen for drugs that regulate LAPTM4B from the entire DNA-mRNA-protein process, reflecting endogenous and diversified cellular regulation.
[0102] (3) Results Analysis
[0103] Comparative experiments show that the system of the present invention significantly improves the screening accuracy and specificity, and can reflect the real regulatory changes of LAPTM4B under physiological conditions.
[0104] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.
Claims
1. A drug for increasing the expression of lysosome-associated transmembrane protein 4B, characterized in that, The drug is selected from one or more of Ruboxistaurin, Motesanib, SGC0946, GSK-461364, Imatinib, and Gefitinib.
2. Application of Ruboxistaurin, Motesanib, SGC0946, GSK-461364, Imatinib, or Gefitinib in the preparation of products that enhance the expression of lysosome-associated transmembrane protein 4B.
3. The application of drugs that enhance the expression of lysosome-associated transmembrane protein 4B in the preparation of cancer models, characterized in that, The drug is selected from one or more of Ruboxistaurin, Motesanib, SGC0946, GSK-461364, Imatinib, and Gefitinib.
4. The application according to claim 3, characterized in that, The tumors include hepatocellular carcinoma, breast cancer, lung cancer, stomach cancer, or ovarian cancer.
5. The application of a drug that enhances the expression of lysosome-associated transmembrane protein 4B in the preparation of lysosome-associated transmembrane protein 4B modulators or vaccine adjuvants, characterized in that, The drug that enhances the expression of lysosome-associated transmembrane protein 4B is selected from one or more of Ruboxistaurin, Motesanib, SGC0946, GSK-461364, Imatinib, and Gefitinib.
6. A reagent for promoting tumor cell proliferation and survival, characterized in that, The reagent is selected from one or more of Ruboxistaurin, Motesanib, SGC0946, GSK-461364, Imatinib, and Gefitinib.
7. A method for screening drugs that induce drug resistance in tumor cells, characterized in that, The method involves administering a drug that enhances the expression of lysosome-associated transmembrane protein 4B to tumor cells, followed by the addition of a drug to be screened. The drug that enhances the expression of lysosome-associated transmembrane protein 4B is selected from one or more of Ruboxistaurin, Motesanib, SGC0946, GSK-461364, Imatinib, and Gefitinib.
8. A method for screening drugs that enhance the expression of lysosome-associated transmembrane protein 4B, characterized in that: Includes the following steps: S1. Construct recombinant cells expressing lysosome-associated transmembrane protein 4B and lysosome-associated membrane protein 3, wherein the lysosome-associated transmembrane protein 4B is labeled with a first fluorescent protein, the lysosome-associated membrane protein 3 is labeled with a second fluorescent protein, and the first fluorescent protein and the second fluorescent protein are different. S2. Co-incubate the drug to be screened with recombinant cells and detect the fluorescence signal. Calculate the ratio of the fluorescence signal of the first fluorescent protein to the fluorescence signal of the second fluorescent protein in the recombinant cells to screen for drugs that enhance the expression of lysosome-associated transmembrane protein 4B.
9. The screening method according to claim 8, characterized in that: In step S1, the first fluorescent protein-labeled lysosome-associated transmembrane protein 4B is obtained by inserting the first fluorescent protein between the 847th and 848th positions of the gene sequence of lysosome-associated transmembrane protein 4B. The second fluorescent protein-labeled lysosome-associated membrane protein 3 is obtained by inserting the second fluorescent protein between positions 786 and 787 of the gene sequence of lysosome-associated membrane protein 3.
10. The screening method according to claim 8, characterized in that: In step S1, the recombinant cells are prepared by introducing the Cas9 protein-coding gene, sgRNA pairs, and donor DNA into recipient cells. The sequences of the sgRNA pairs are shown in SEQ ID NO.1-4, the gene sequences of the donor DNA are shown in SEQ ID NO.5-6, and the recipient cells express lysosome-associated transmembrane protein 4B and lysosome-associated membrane protein 3.