Application of METTL1 overexpression agent in preparation of anti-mycobacterium tuberculosis drugs

Abstract

The invention discloses an application of an METTL1 overexpression agent in preparation of an anti-mycobacterium tuberculosis medicine, the METTL1 overexpression agent is an METTL1 overexpression lentivirus, the registration number of the sequence of the METTL1 overexpression lentivirus in an NCBI database is NM005371.6, and the anti-mycobacterium tuberculosis medicine is capable of improving the autophagy level of mycobacterium tuberculosis infected macrophages, and has the advantages that the anti-mycobacterium tuberculosis medicine can be used for preparing the anti-mycobacterium tuberculosis medicine; a drug for enhancing the ability of macrophages to remove intracellular Mycobacterium tuberculosis; the METTL1 overexpression agent provided by the invention is proved to be capable of overexpressing METTL1 genes through cell experiments, and has remarkable effects in multiple aspects of promoting autophagy of mycobacterium tuberculosis infected macrophages, enhancing bactericidal ability and the like, and a new targeted therapeutic drug is provided for clinical treatment of mycobacterium tuberculosis infection resistance.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to application of a METTL1 overexpression agent in the preparation of an anti-Mycobacterium tuberculosis drug. Background Art

[0002] Tuberculosis (TB), a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb), remains a serious threat to human health. According to the World Health Organization (WHO), the estimated global TB incidence in 2023 is 10.8 million, with an incidence rate of 134 cases per 100,000 people. The global TB death toll is expected to reach 1.25 million in 2023, making TB the leading single infectious cause of death globally, with a death toll nearly double that of HIV / AIDS. my country has an estimated TB incidence of 741,000 cases and an incidence rate of 52 cases per 100,000 people, ranking third among countries with a high TB burden and one of the highest rates of multidrug-resistant TB. my country also has a large population of people with latent TB infection (LTBI). The estimated LTBI rate among people aged 5 and above is 18.1%. Due to its unique latent nature and the emergence and spread of drug-resistant tuberculosis, tuberculosis prevention and control faces enormous challenges. Therefore, discovering new targets for anti-tuberculosis infection and developing new anti-tuberculosis drugs have become the most concerned issues.

[0003] Mycobacterium tuberculosis is a typical intracellular parasite that primarily resides within macrophages after infecting the body. Macrophages are a type of immune cell widely present in various tissues and body fluids of the human body. They possess a powerful phagocytic capacity and serve as the body's first line of defense against invasion by M. tuberculosis. On the one hand, after infection, M. tuberculosis's proteins, phospholipids, and other antigens are recognized by multiple macrophage receptors, triggering corresponding antimicrobial responses in the macrophages, such as clearance through autophagy, apoptosis, and the production of free radicals (ROS and RNS). On the other hand, M. tuberculosis has evolved complex immune escape strategies to counteract autophagic killing. For example, after being engulfed into the macrophage phagosome, M. tuberculosis can successfully prevent the fusion of the phagosome with the lysosome, inhibiting the maturation of the phagolysosome and thereby preventing its own degradation by the lysosome. Furthermore, M. tuberculosis can also use macrophages as a shelter, allowing it to remain dormant and even proliferate.

[0004] Studies have shown that autophagy plays a crucial role in macrophages' resistance to Mycobacterium tuberculosis infection, and enhancing macrophage autophagy can reduce the survival of Mycobacterium tuberculosis within cells. The primary mechanism may be that autophagy activation increases the colocalization of autophagic proteins with phagosomes, promoting the transformation of phagosomes containing Mycobacterium tuberculosis into mature phagolysosomes, and thereby promoting the clearance of Mycobacterium tuberculosis within macrophages through the autophagolysosomal pathway. Identifying and analyzing the functional genes involved in the invasion of Mycobacterium tuberculosis into host macrophages, particularly those regulating macrophage autophagy, could provide important target sites for the design and screening of anti-tuberculosis drugs.

[0005] N 7 -methylguanosine (N 7 -methylguanosine, m 7 G) is a ubiquitous and evolutionarily conserved RNA methylation modification. 7 G widely affects mRNA stability, translation fidelity and non-coding RNA function by regulating RNA splicing, nucleocytoplasmic transport and translation initiation complex assembly. Methyltransferase-like 1 (METTL1) is a classic m 7 G methyltransferase, which forms a stable methyltransferase complex with WDR4 and specifically catalyzes guanosine N 7 Studies have shown that METTL1 can modify the methylation of 7 The G modification pathway affects the mRNA stability and translation efficiency of target genes, regulates target gene expression, and plays an important role in the occurrence and development of various human diseases including malignant tumors, cardiovascular diseases, and infectious diseases. However, at present, the relevant application of the METTL1 gene in the treatment of tuberculosis has not been disclosed. Summary of the Invention

[0006] In view of the problems existing in the prior art, the purpose of the present invention is to provide the application of a METTL1 overexpression agent in tuberculosis. The inventors have found for the first time that the expression of METTL1 in peripheral blood mononuclear cells of patients with pulmonary tuberculosis is significantly downregulated. The present invention designs and synthesizes a reagent that specifically overexpresses METTL1 by utilizing lentiviral vector technology. The above-mentioned METTL1 overexpression agent can specifically promote the expression of METTL1, thereby promoting the autophagy level of macrophages infected with Mycobacterium tuberculosis and enhancing the clearance of intracellular Mycobacterium tuberculosis by macrophages. The present invention can be used for the development of tuberculosis treatment reagents by constructing a METTL1 overexpression agent. METTL1 overexpression agents will play an important role in the fields of gene therapy and molecular targeted therapy for tuberculosis, and provide new targeted therapeutic drugs for the clinical treatment of tuberculosis.

[0007] In order to achieve the above objectives, the present invention adopts the following technical solutions.

[0008] A METTL1 overexpression agent was constructed using a lentiviral vector and the METTL1 gene.

[0009] According to a first aspect of the present invention, a METTL1 overexpression agent is provided. The METTL1 overexpression agent is a METTL1 overexpression lentivirus. The accession number of the sequence of the METTL1 overexpression lentivirus in the NCBI database is NM_005371.6.

[0010] In some embodiments, the METTL1 overexpression agent comprises the nucleotide sequence shown in SEQ ID NO.1, which is: - (SEQ ID NO. 1) According to a second aspect of the present invention, there is provided a use of a METTL1 overexpression agent in the preparation of an anti-Mycobacterium tuberculosis drug.

[0011] In some embodiments, the anti-Mycobacterium tuberculosis drug refers to a drug that can increase the autophagy level of macrophages infected with Mycobacterium tuberculosis and enhance the ability of macrophages to clear intracellular Mycobacterium tuberculosis.

[0012] In some embodiments, the drug uses a METTL1 overexpression agent as an active ingredient and is prepared into a pharmaceutically acceptable dosage form using a pharmaceutically acceptable carrier.

[0013] In some embodiments, the carrier is solid, liquid, or semisolid.

[0014] In some embodiments, the dosage forms include tablets, capsules, injections, and drops.

[0015] The beneficial effects of the present invention are: 1. This invention proposes for the first time the use of lentiviral vector technology to develop a reagent that targets overexpression of the METTL1 gene. This reagent can significantly promote autophagy in macrophages infected with Mycobacterium tuberculosis and enhance macrophage clearance of intracellular Mycobacterium tuberculosis. It represents a new targeted drug formulation for the treatment of tuberculosis and possesses significant specificity compared to traditional therapeutic drugs.

[0016] 2. The METTL1 overexpression agent provided by the present invention has been confirmed through experiments to be able to effectively promote the expression of the METTL1 gene, and has significant effects in promoting the autophagy level of macrophages infected with Mycobacterium tuberculosis and enhancing the clearance of intracellular Mycobacterium tuberculosis by macrophages. It achieves the purpose of gene therapy for tuberculosis and provides a new targeted therapeutic drug for the clinical treatment of tuberculosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 The expression levels of METTL1 in peripheral blood mononuclear cells (PBMCs) of the pulmonary tuberculosis patient group and the healthy control group in Example 1 of the present invention are shown in Figure A, wherein Figure A shows the mRNA expression level of METTL1 in each group detected by RT-qPCR, and Figure B shows the protein expression level of METTL1 in each group detected by Western blot; Figure 2 The expression of METTL1 in macrophages in pulmonary tuberculosis granulomas and control lung tissues of pulmonary tuberculosis patients in Example 1 of the present invention; Figure 3Figure 2 shows the expression of METTL1 in THP-1 cells treated with a METTL1 overexpression agent (oe-METTL1) and a blank control group (oe-NC) in Example 2 of the present invention. Figure A shows the mRNA expression level of METTL1 in each group detected by RT-qPCR, and Figure B shows the protein expression level of METTL1 in each group detected by Western blot. Figure 4 The Western blot analysis of Example 3 of the present invention detected the effect of METTL1 on the autophagy function of macrophages infected with Mycobacterium tuberculosis; Figure 5 The effect of METTL1 on the autophagy function of macrophages infected with Mycobacterium tuberculosis was detected by cell immunofluorescence in Example 3 of the present invention; Figure 6 The effect of METTL1 on the autophagy function of macrophages infected with Mycobacterium tuberculosis detected by transmission electron microscopy in Example 3 of the present invention; Figure 7 The colony forming unit (CFU) experiment in Example 3 of the present invention was used to detect the effect of METTL1 on macrophage clearance of intracellular Mycobacterium tuberculosis; Figure 1-Figure 7 There were statistically significant differences in the changes among the groups. DETAILED DESCRIPTION

[0019] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0020] The present invention will be further described in detail below with reference to the embodiments.

[0021] Example 1: Analysis of METTL1 expression in patients with active pulmonary tuberculosis 1. Objects and Methods 1. Participants: 53 patients with active pulmonary tuberculosis admitted to Jiangxi Provincial Chest Hospital and the First Affiliated Hospital of Nanchang University. The diagnosis of active pulmonary tuberculosis was based on the diagnostic criteria for pulmonary tuberculosis (WS288-2017) published by the National Health Commission. All patients with active pulmonary tuberculosis had not received any anti-tuberculosis treatment and had positive results for Mycobacterium tuberculosis by liquid culture and sputum smear acid-fast staining. Fifty healthy controls were recruited from the Health Management Center of the First Affiliated Hospital of Nanchang University. The control group was matched by age (±3 years), sex (1:1 matching), and body mass index (±10%). Chest radiographs showed no abnormalities, no recent history of contact with tuberculosis, and a negative T-SPOT test result. Patients with concomitant cancer, hypertension, diabetes, autoimmune diseases, or taking immunosuppressive medications were excluded. Patients with HBV, HCV, HIV infection, or / and AIDS were also excluded. Pregnant or lactating women were also excluded.

[0022] 2. Methods (1) Extraction of peripheral blood mononuclear cells (PBMC): In the morning, 5 mL of fasting venous blood was collected from the subjects and anticoagulated with EDTA-K2. The anticoagulated blood was thoroughly mixed with an equal volume of Hanks' solution to prepare a mixture. In a separate centrifuge tube, an equal volume of lymphocyte separation solution was added. Using a pipette, the mixture was slowly pipetted onto the surface of the lymphocyte separation solution along the tube wall, keeping the liquid surface clear. The tube was centrifuged at 2000 rpm for 20 minutes. The intermediate buffy coat layer was carefully aspirated and transferred to a new centrifuge tube. Five volumes of Hanks' solution were added, and the cells were washed twice by centrifugation at 1500 rpm for 10 minutes. The supernatant was removed from the final centrifugation, and the cells were resuspended in RPMI1640 containing 10% (v / v) fetal bovine serum. 10 μL of the suspension was collected from each subject for counting.

[0023] (2) Extraction of total RNA: Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) of the research subjects using the Trizol method: 500 μL of TRIzol lysis buffer was added to the extracted PBMCs and vortexed immediately; after standing for 5 minutes, 200 μL of chloroform was immediately added and vortexed to mix thoroughly, and the tube was allowed to stand for 10 minutes; the tube was centrifuged at 16,000 g for 20 minutes at 4°C; 450 μL of supernatant was added to 800 μL of isopropanol and gently inverted to mix thoroughly, and the tube was precipitated overnight; after centrifugation at 16,000 g for 20 minutes at 4°C, the supernatant was discarded, and 1 mL of 75% ethanol was added and gently shaken to wash; after centrifugation at 16,000 g for 15 minutes at 4°C, the supernatant was discarded, 500 μL of 75% anhydrous ethanol (prepared with DEPC water) was added and gently mixed, and the tube was centrifuged at 10,000 g for 5 minutes at 4°C, the supernatant was discarded, and the tube was inverted on absorbent paper to dry for 10 minutes; RNA was dissolved in 30 μL of DEPC water for later use.

[0024] (3) RT-qPCR detection of mRNA expression: 1) Quantify METTL1 mRNA expression in each group using real-time fluorescence quantitative PCR. Total RNA was extracted from PBMCs in each group using the Trizol method. The purity and concentration of the total RNA were determined and set aside.

[0025] 2) Synthesis of template DNA, i.e., cDNA: Reverse transcribe the prepared total RNA according to the instructions of the Takara reverse transcription reaction kit. The amplified product is the template cDNA and is stored at -20°C until use.

[0026] 3) SYBR Green RT-qPCR detection: ①METTL1 primers and internal reference gene GAPDH primers were synthesized by BGI and dissolved in nuclease-free water to 10 nmol / mL for later use; ② Prepare the RT-qPCR reaction system using the Takara fluorescence quantitative kit as shown in the following table: Element Dosage SYBR® Premix Ex TaqTM II (2×) 10.0μL Forward Primer (10 μM) 1.0 μL Reverse Primer (10 μM) 1.0 μL ROX Reference Dye (50×) 0.4μL cDNA 2.0 μL ddH2O Add to 20 μL ③ Reaction conditions: first 95℃ for 30s, then 40 cycles of 95℃ for 5s, 60℃ for 34s, and finally 95℃ for 15s, 60℃ for 60s, and 95℃ for 15s; ④ To reduce experimental errors, each sample was tested three times.

[0027] ⑤ Analyze the results: The amplification effect was determined by combining the amplification curve and the melting curve, and the expression level of the target gene was calculated using the 2-ΔΔCt method.

[0028] (4) Western blot detection of protein expression: Cells from each group were collected and washed with PBS. Total cell protein was extracted by RIPA lysis method, and total protein concentration was determined by BCA method. 40 μg of protein was dissolved in 6×SDS loading buffer and boiled at 100℃ for 10 min. After that, each sample was subjected to 10% SDS-PAGE gel electrophoresis. The protein was transferred to PVDF membrane by wet transfer method and blocked with 5% skim milk powder at room temperature for 2 h. The PVDF membrane was then incubated with 1:1000 diluted primary antibodies METTL1 and β-actin at 4℃ overnight. After washing with TBST three times, the membrane was incubated with 1:5000 diluted rabbit secondary antibody at room temperature for 1 h. After rinsing with TBST, developer was added and imaging was performed using Bio-Rad Image Analysis System.

[0029] (5) Fluorescent immunohistochemistry to detect intracellular gene expression: Prepare frozen sections, place the sections in environmentally friendly dewaxing solution for 10 minutes, wash three times with anhydrous ethanol for 5 minutes, and then wash with distilled water. After the sections are cooled naturally, place the slides in PBS and wash them on a decolorizing shaker for 5 minutes, repeating three times. Place the sections in 3% hydrogen peroxide solution and incubate them at room temperature in the dark for 25 minutes. Place the slides in PBS and wash them on a decolorizing shaker for 5 minutes, repeating three times. Block with 3% BSA at room temperature for 30 minutes. Shake off the blocking solution, add diluted primary antibody to the sections, and incubate them flat in a humidified box at 4°C overnight. After washing, add fluorescent secondary antibody and incubate at room temperature for 50 minutes (repeat the above steps according to the required number of multiple targets). After incubation with DAPI, use mounting medium to seal the sections and scan the entire section under a fluorescence confocal microscope.

[0030] 2. Results The applicant of the present invention tested peripheral blood PBMC samples from 53 tuberculosis patients (TB) and 50 healthy controls (HC). The results showed that the mRNA level of METTL1 in PBMC of the TB group was significantly downregulated compared with that of the healthy control group, and the difference was statistically significant ( p <0.0001, Figure 1 A). The protein level of METTL1 in peripheral blood PBMCs of the pulmonary tuberculosis group was significantly downregulated compared with that of the healthy control group ( Figure 1 B). Fluorescence immunoassay was used to detect the expression of METTL1 in macrophages in pulmonary tuberculosis granulomas and control lung tissues. Figure 2 As shown, the expression of METTL1 in macrophages in tuberculosis granuloma tissues was reduced compared with that in control lung tissues.

[0031] Example 2: Design, preparation and validation of a METTL1 overexpression agent.

[0032] 1. Methods 1. Design an overexpression agent for METTL1. The target sequence is the CDS sequence of the METTL1 gene in the NCBI database: - (SEQ ID NO. 1) Based on the above target sequence, a plasmid for targeted overexpression of METTL1 gene was synthesized and further packaged into oe-METTL1 lentivirus for cell, animal and therapeutic experiments.

[0033] 2. Transfect cells with lentivirus to overexpress METTL1. THP-1 cells were cultured in complete medium with 10% serum concentration at 37°C in a 5% CO2 incubator. Cells in logarithmic growth phase were taken and replaced with serum-free medium. Cells were resuspended to a concentration of 10 5 Cells transfected with oe-METTL1 lentivirus were designated as the oe-METTL1 experimental group, and cells treated with empty plasmid lentivirus were designated as the oe-NC blank control group.

[0034] 3. RT-qPCR was used to detect the mRNA expression of the METTL1 gene in each group of cells using the same method as described above.

[0035] 4. Western Blot was used to detect the expression of METTL1 gene protein in each group of cells using the same method as described above.

[0036] 2. Results The results showed that the expression level of METTL1 mRNA in the oe-METTL1 group was significantly higher than that in the oe-NC group ( p =0.0004, Figure 3 A); The expression level of METTL1 protein in the oe-METTL1 group was significantly higher than that in the oe-NC group ( Figure 3 B).

[0037] Example 3: Effects of METTL1 on autophagy and bactericidal function of human macrophages infected with Mycobacterium tuberculosis 1. Methods 1. Establishment of an in vitro Mycobacterium tuberculosis infection model (1) Activation of macrophages: Take THP-1 cells in the logarithmic growth phase, centrifuge and remove the supernatant, resuspend the cells in THP-1 complete medium, and culture THP-1 at 6×10 5 Inoculate 100 cells / well in a 25 mm dish, plate four dishes in parallel, add PMA (50 ng / mL), mix gently, and incubate in a 37°C, 5% CO2 incubator for 24 h. Replace the culture medium and continue incubation for another 24 h.

[0038] (2) Establishment of an in vitro infection model at different MOIs: Add Mycobacterium tuberculosis H37Ra suspension to activated THP-1 macrophages at MOIs of 0, 1, 5, and 10, respectively, and culture in a 37°C, 5% CO2 incubator for 4 h. Discard the old culture medium, wash the cells three times with 1× PBS, add fresh THP-1 macrophage complete culture medium, and continue culturing for 24 h. Cell RNA and protein are collected.

[0039] (3) Establishment of in vitro models at different time periods after infection: H37Ra suspension was added to activated THP-1 macrophages at an MOI of 10 and cultured in a 37°C, 5% CO2 incubator for 4 h. The old culture medium was discarded, the cells were washed three times with 1× PBS, and fresh THP-1 macrophage complete culture medium was added. The cells were cultured for 0 h, 4 h, 24 h, 48 h, and 72 h, and the cellular RNA and protein were collected.

[0040] 2. Colony Forming Unit (CFU) Assay The THP-1 macrophages treated as above were lysed with 0.03% SDS, and the lysate was then diluted serially. 100 μL of the diluted bacterial solution was inoculated on a 7H10 agar plate and cultured in a 37°C, 5% CO2 incubator for 3-4 weeks. The results were observed and the colonies were counted.

[0041] 3. Detection of Macrophage Autophagy Indicators The above macrophages were obtained and the expression level of the autophagy marker LC3B was detected by Western blot using the same method as described above.

[0042] The above macrophages were obtained and the expression level of the autophagy marker LC3B was detected by cell immunofluorescence using the same method as described above.

[0043] 4. Detection of Autophagy Levels by Transmission Electron Microscopy The cells were collected by centrifugation, fixed with 2.5% glutaraldehyde solution overnight, washed with PBS three times, 15 minutes each time, and then embedded in 1% agarose solution. The cells were fixed with 1% osmium acid at room temperature in the dark for 3 hours, washed with PBS three times, 15 minutes each time, and dehydrated in 50% ethanol-70% ethanol-90% ethanol-90% ethanol:90% acetone (1:1)-90% acetone for 15 minutes. The cells were embedded in a solution of pure acetone and embedding solution at a ratio of 2:1 at room temperature for 3 minutes. h, then replaced with a 1:2 ratio mixture, and continued embedding at room temperature overnight. Finally, pure embedding solution was used for embedding at room temperature for 3 h, and cured in an oven at 37°C, 45°C, and 60°C overnight, 12 h, and 48 h, respectively. After slicing, the sections were stained with 3% uranyl acetate saturated ethanol in the dark for 8 min, washed 3 times with 70% ethanol, washed 3 times with ddH2O, stained in 2.6% lead citrate solution in the dark for 8 min, and finally washed 3 times with ddH2O. After drying at room temperature overnight, autophagosomes and autolysosomes were observed under a transmission electron microscope.

[0044] 2. Results LC3B is a marker of the autophagy process. Monitoring the expression level of LC3B can help us understand the development and changes of autophagy in the body. It also shows the effect of METTL1 on the autophagic function of macrophages after infection with Mycobacterium tuberculosis. Western blot analysis of LC3B protein showed that when not infected with Mycobacterium tuberculosis (CON), the LC3B protein level in the METTL1 gene overexpression group (oe-METTL1) was significantly higher than that in the oe-NC group. After infection with Mycobacterium tuberculosis (24h), the LC3B protein expression levels in both the oe-METTL1 and oe-NC groups increased further, and the LC3B expression level in the oe-METTL1 group was significantly higher than that in the oe-NC group ( Figure 4 The applicant of the present invention found that Mycobacterium tuberculosis infection can reduce the expression level of METTL1 in macrophages, and high expression of METTL1 can promote autophagy in macrophages infected with Mycobacterium tuberculosis, while low expression of METTL1 inhibits autophagy in macrophages infected with Mycobacterium tuberculosis ( Figure 4 ).

[0045] The METTL1 gene-stably overexpressing THP-1 macrophages oe-METTL1 and the control stable transgenic strain oe-NC were used to construct an in vitro macrophage model of Mycobacterium tuberculosis infection. The production of LC3B was further detected by cell immunofluorescence. The results showed that after infection with Mycobacterium tuberculosis, the LC3B protein level in the oe-METTL1 group increased compared with the oe-NC group ( p =0.0009, Figure 5 ), indicating that METTL1 overexpression can promote autophagy in macrophages infected with Mycobacterium tuberculosis.

[0046] Transmission electron microscopy is the gold standard for monitoring the autophagy process. We used THP-1 macrophages stably overexpressing the METTL1 gene, oe-METTL1, and a control stably overexpressing strain, oe-NC, to establish an in vitro macrophage infection model with Mycobacterium tuberculosis. Transmission electron microscopy was used to examine the autophagy level in macrophages. The results showed that after infection with Mycobacterium tuberculosis, the accumulation of autophagic cells and autolysosomes in the oe-METTL1 group was greater than that in the oe-NC group ( Figure 6 ), indicating that METTL1 overexpression can promote autophagy in macrophages infected with Mycobacterium tuberculosis.

[0047] In the above experiments, the present applicants discovered that overexpression of METTL1 promoted autophagy in macrophages infected with Mycobacterium tuberculosis. Therefore, the present applicants used the oe-METTL1 stable transfectant and the control stable transfectant oe-NC to construct an in vitro macrophage infection model with Mycobacterium tuberculosis. Cell lysates were collected from uninfected and infected cells 48 hours after plating for culture. The results showed that as the post-infection period increased, the number of surviving bacteria in the oe-METTL1 group was significantly lower than that in the oe-NC group ( p =0.0015, Figure 7 ), indicating that overexpression of METTL1 enhanced the clearance of intracellular Mycobacterium tuberculosis by macrophages and promoted macrophage bactericidal killing.

[0048] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the creative concept of the present invention, which all fall within the scope of protection of the present invention.

Claims

1. A METTL1 overexpression agent, characterized in that The METTL1 overexpression agent is a METTL1 overexpression lentivirus, and the accession number of the METTL1 overexpression lentivirus sequence in the NCBI database is NM_005371.

6.

2. The METTL1 overexpression agent according to claim 1, characterized in that The METTL1 overexpression agent includes the nucleotide sequence shown in SEQ ID NO.

1.

3. Use of the METTL1 overexpression agent according to claim 1 or 2 in the preparation of an anti-Mycobacterium tuberculosis drug.

4. The use according to claim 3, characterized in that The anti-Mycobacterium tuberculosis drug refers to a drug that can increase the autophagy level of macrophages infected with Mycobacterium tuberculosis and enhance the ability of macrophages to clear intracellular Mycobacterium tuberculosis.

5. The use according to claim 3, characterized in that The drug uses a METTL1 overexpression agent as an active ingredient and is prepared into a pharmaceutically acceptable dosage form using a pharmaceutically acceptable carrier.

6. The use according to claim 5, characterized in that The carrier is solid, liquid or semi-solid.

7. The use according to claim 5, characterized in that The dosage forms include tablets, capsules, injections and drops.

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