A medicine for resisting tuberculosis infection and application thereof

Abstract

The application discloses application of SLC44A1 in preparation of anti-tuberculosis drugs and belongs to the technical field of biotechnology. It is first determined that the solute carrier family 44 member 1 protein SLC44A1 can significantly inhibit the proliferation of mycobacterium tuberculosis in macrophages. The SLC44A1 can be used as a new potential drug for resisting tuberculosis infection. In addition, the SLC44A1 can be combined with other active substances for promoting the anti-tuberculosis activity of the body and / or drugs for improving the symptoms of tuberculosis to form a combined drug, so that a new treatment method and drug for resisting tuberculosis infection are provided.

Description

A drug for treating tuberculosis infection and its application Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the discovery of drugs against Mycobacterium tuberculosis infection by screening important target sites for anti-tuberculosis drugs. Background Technology

[0002] Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis (M.tb). Although the current mainstream strategy for tuberculosis treatment relies on anti-tuberculosis drugs with bactericidal or bacteriostatic functions, the success rate of traditional anti-tuberculosis treatment is greatly affected by the emergence of drug-resistant tuberculosis patients, the existence of serious adverse reactions of anti-tuberculosis drugs, and poor compliance due to long treatment cycles. Current research suggests that host-guided anti-tuberculosis immunotherapy can target highly conserved host signaling pathways, improve the success rate of treatment on the basis of conventional anti-tuberculosis treatment, and even shorten the treatment time, especially in the treatment of multidrug-resistant tuberculosis and extensively drug-resistant tuberculosis, which can bring better treatment results (Guo Xueying et al. (2023) Chinese Journal of Zoonoses, 2023, 39(11):1124-1129.). Using molecular biology techniques to screen and identify genes or proteins involved in resistance to M.tb infection and pathogenesis can provide important drug targets for the design and screening of novel tuberculosis drugs, which has significant theoretical and practical value for the comprehensive prevention and control of tuberculosis.

[0003] Mycobacterium tuberculosis (M.tb) is a typical intracellular bacterium that causes tuberculosis. After infecting the body, M.tb primarily parasitizes macrophages. The macrophage-mediated innate immune response is the host's first line of defense against tuberculosis infection, and the struggle between macrophages and M.tb determines the outcome of the infection. M.tb has evolved highly efficient and complex methods to evade or interfere with host immune signaling pathways, including preventing the maturation of phagocytosomial-lysosome systems within macrophages, inducing the expression of anti-inflammatory factors, disrupting macrophage pattern recognition receptors, and inhibiting macrophage aggregation. Therefore, in-depth research into the molecular mechanisms of intracellular survival of Mycobacterium tuberculosis is crucial for providing new targets for the development of anti-tuberculosis drugs. Identifying genes or proteins involved in resisting M.tb infection and pathogenesis, and developing corresponding therapeutic drugs targeting these genes or proteins, will be an important approach to achieving host-guided immunotherapy for tuberculosis.

[0004] Solute carriers (SLCs) are a family of membrane transport proteins, comprising over 300 members, most of which are located on the cell membrane. Their primary function is to facilitate the transport of various substrates across biological membranes, including the uptake of small molecules by cells. SLC44 family transport proteins have become promising new targets in the treatment and diagnosis of immune and degenerative diseases. SLC44A1, as a choline carrier, participates in choline transport and transmembrane transport, and is widely expressed in neurons and dendritic cells throughout the nervous system. It can be detected in both the plasma and mitochondrial membranes, and therefore participates in the regulation of proliferation in some diseases, such as lung cancer cell lines (Traiffort E et al. (2013) Mol Aspects Med. 10.1016 / j.mam.2012.10.011.). Driven by host-guided anti-tuberculosis treatment strategies, research on target sites aims to discover new drugs with anti-tuberculosis effects. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention discovered a solute carrier family 44 member 1 (SLC44A1) that can significantly regulate and inhibit the proliferation of Mycobacterium tuberculosis in host macrophages and lung tissue, and based on this, the invention was completed.

[0006] The technical solution of this invention is as follows:

[0007] A first aspect of the present invention provides the use of SLC44A1 in the preparation of a medicament against Mycobacterium tuberculosis infection, wherein the amino acid sequence of SLC44A1 is shown in SEQ ID NO.1, wherein SLC44A1 is a functional protein of solute carrier family 44 member 1, present in the cytoplasm and mitochondria, and has a significant regulatory and inhibitory effect on the proliferation of Mycobacterium tuberculosis in host macrophages. SEQ ID NO.1:

[0008] 1 mgccssassa aqsskrewkp ledrsctdip wlllfilfci gmgficgfsi atgaaarlvs

[0009] 61 gydsygnicg qkntkleaip nsgmdhtqrk yvffldpcnl dlinrkiksv alcvaacprq

[0010] 121 elktlsdvqk faeingsalc synlkpseyt tspkssvlcp klpvpasapi pffhrcapvn

[0011] 181 iscyakfaea litfvsdnsv lhrlisgvmt skeiilglcl lslvlsmilm viiryisrvl

[0012] 241 vwiltilvil gslggtgvlw wlyakqrrsp ketvtpeqlq iaednlrall iyaisatvft

[0013] 301 vilflimlvm rkrvaltial fhvagkvfih lpllvfqpfw tffalvlfwv ywimtllflg

[0014] 361 ttgspvqneq gfvefkisgp lqymwwyhvv gliwisefil acqqmtvaga vvtyyftrdk

[0015] 421 rnlpftpila svnrliryhl gtvakgsfii tlvkiprmil myihsqlkgk enacarcvlk

[0016] 481 scicclwcle kclnylnqna ytatainstn fctsakdafv ilvenalrva tintvgdfml

[0017] 541 flgkvlivcs tglagimlln yqqdytvwvl pliivclfaf lvahcflsiy emvvdvlflc

[0018] 601 faidtkyndg spgrefymdk vlmefvensr kamkeagkgg vadsrelkpm lkkr

[0019] Furthermore, the amino acid sequence of the SLC44A1 protein also includes 80%-99% homologous sequence, preferably 80%-85%; preferably 85%-90%; preferably 90%-95% homologous sequence.

[0020] Furthermore, the amino acid sequence of the SLC44A1 protein has an amino acid deletion, mutation, or addition at one or more sites.

[0021] Furthermore, the SLC44A1 protein also includes its fusion protein, conjugate, nucleic acid encoding its fusion protein or conjugate, and vector expressing the aforementioned nucleic acid molecules.

[0022] Furthermore, the aforementioned Mycobacterium tuberculosis infection includes: primary infection, secondary infection, and extrapulmonary infection.

[0023] Furthermore, the tuberculosis mentioned includes, but is not limited to, drug-resistant tuberculosis, non-drug-resistant tuberculosis, pulmonary tuberculosis, and extrapulmonary tuberculosis.

[0024] Furthermore, the drug-resistant tuberculosis mentioned includes, but is not limited to, single-drug resistant tuberculosis, multidrug resistant tuberculosis, multidrug-resistant tuberculosis, and extensively drug-resistant tuberculosis.

[0025] Furthermore, the pulmonary tuberculosis mentioned includes primary pulmonary tuberculosis, secondary pulmonary tuberculosis, hematogenous disseminated pulmonary tuberculosis, tracheobronchial tuberculosis, tuberculous pleurisy, and sputum-negative pulmonary tuberculosis.

[0026] Furthermore, the extrapulmonary tuberculosis includes, but is not limited to, lymph node tuberculosis, intestinal tuberculosis, renal tuberculosis, and bone and joint tuberculosis.

[0027] Furthermore, the Mycobacterium tuberculosis includes multidrug-resistant Mycobacterium tuberculosis and extensively drug-resistant Mycobacterium tuberculosis.

[0028] Furthermore, the tuberculosis mycobacteria include Mycobacterium humanis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium cannerae, and Mycobacterium villiformis.

[0029] Furthermore, the hosts infected by the Mycobacterium tuberculosis include humans, cattle, rats, badgers, deer, primates, etc.

[0030] Furthermore, the drug has at least one of the following effects:

[0031] Inhibits the activity of Mycobacterium tuberculosis;

[0032] Anti-tuberculosis mycobacterial infection;

[0033] Prevention and / or treatment of diseases caused by Mycobacterium tuberculosis;

[0034] Furthermore, the drug includes at least one of a pharmaceutical carrier, excipient, and excipient.

[0035] Furthermore, the excipients include at least one of the following: excipients, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, penetration promoters, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, antioxidants, adsorbents, filter aids, and release inhibitors.

[0036] Furthermore, the pharmaceutical preparation includes biological agents or pharmaceutical preparations.

[0037] Furthermore, the dosage forms of the anti-tuberculosis drugs include: sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquids, lozenges, granules, powders, pills, suspensions, powders, tinctures, preparations, drops, injections, powder for injection, creams, sustained-release preparations, targeted agents, etc.

[0038] Furthermore, the administration methods of the anti-tuberculosis drug preparation include oral, injection, implantation, external application, spraying, and inhalation.

[0039] Furthermore, the drug can be delivered into the host body via delivery systems such as gene delivery devices, AVVs, and liposomes to complete in vivo treatment.

[0040] In a second aspect, the present invention provides a pharmaceutical composition for combating Mycobacterium tuberculosis, the pharmaceutical composition comprising SLC44A1 and another drug for combating Mycobacterium tuberculosis, wherein the amino acid sequence of SLC44A1 is shown in SEQ ID NO.1. SLC44A1 is a functional protein of solute carrier family 44 member 1 present in the cytoplasm and mitochondria, which has a significant effect on regulating and inhibiting the proliferation of Mycobacterium tuberculosis in host macrophages.

[0041] SEQ ID NO.1:

[0042] 1 mgccssassa aqsskrewkp ledrsctdip wlllfilfci gmgficgfsi atgaaarlvs

[0043] 61 gydsygnicg qkntkleaip nsgmdhtqrk yvffldpcnl dlinrkiksv alcvaacprq

[0044] 121 elktlsdvqk faeingsalc synlkpseyt tspkssvlcp klpvpasapi pffhrcapvn

[0045] 181 iscyakfaea litfvsdnsv lhrlisgvmt skeiilglcl lslvlsmilm viiryisrvl

[0046] 241 vwiltilvil gslggtgvlw wlyakqrrsp ketvtpeqlq iaednlrall iyaisatvft

[0047] 301 vilflimlvm rkrvaltial fhvagkvfih lpllvfqpfw tffalvlfwv ywimtllflg

[0048] 361 ttgspvqneq gfvefkisgp lqymwwyhvv gliwisefil acqqmtvaga vvtyyftrdk

[0049] 421 rnlpftpila svnrliryhl gtvakgsfii tlvkiprmil myihsqlkgk enacarcvlk

[0050] 481 scicclwcle kclnylnqna ytatainstn fctsakdafv ilvenalrva tintvgdfml

[0051] 541 flgkvlivcs tglagimlln yqqdytvwvl pliivclfaf lvahcflsiy emvvdvlflc

[0052] 601 faidtkyndg spgrefymdk vlmefvensr kamkeagkgg vadsrelkpm lkkr

[0053] Furthermore, another type of anti-tuberculosis drug is one that can promote the body's activity against tuberculosis bacteria and / or improve tuberculosis symptoms.

[0054] Furthermore, another active ingredient against Mycobacterium tuberculosis includes, but is not limited to, active ingredients and drugs that act on the cell wall of mycobacteria during the process of fighting tuberculosis infection, active ingredients and drugs that inhibit protein synthesis, active ingredients and drugs that inhibit the activity of ATP synthase, an energy metabolism enzyme, and active ingredients and drugs that inhibit DNA synthesis.

[0055] The beneficial effects of this invention are:

[0056] This invention provides an important target gene or protein for developing host-guided anti-tuberculosis treatment strategies. It is significant for elucidating the mechanism of macrophage-mediated innate immune response in host resistance to tuberculosis infection. Drugs targeting this gene or protein can inhibit the proliferation of Mycobacterium tuberculosis on host macrophages and control the pathogenicity of Mycobacterium tuberculosis. Attached Figure Description

[0057] Figure 1: SLC44A1 expression levels in peripheral blood mononuclear cells (HC: n = 8; LTBI: n = 10; TB: n = 13).

[0058] Figure 2: A. Changes in SLC44A1 expression in THP-1 cells after H37Rv infection (n = 3); B. Relative expression level of SLC44A1 protein in THP-1 cells after H37Rv infection; C. Changes in SLC44A1 expression in human primary macrophages after H37Rv infection (n = 17).

[0059] Figure 3: AB. Comparison of SLC44A1 expression changes in primary macrophages of infected and uninfected mice (n = 3);

[0060] D. Changes in SLC44A1 expression in mouse lung tissue before and after H37Rv infection (n = 3).

[0061] Figure 4: A. Silencer efficiency of SLC44A1 in THP-1 cells; B. CFU detection at 4h, 24h and 48h after H37Rv infection (n=3).

[0062] Figure 5: siRNA inhibition of SLC44A1 leads to a decrease in the proportion of apoptosis in THP-1 cells; A. Illustration of apoptosis in H37Rv-infected cells 24h; B. Proportion of apoptosis in H37Rv-infected and uninfected cells (n = 3).

[0063] Figure 6: A. Detection of caspase-3 protein in SLC44A1 silenced cells and control empty vector cells after H37Rv infection; B. Relative expression level of cleaved caspase-3 protein (n = 5).

[0064] Figure 7: A. Detection of caspase-8 and caspase-9 proteins in H37Rv-infected SLC44A1 silent cells and control cells; B. Relative expression levels of cleaved caspase-8 and cleaved caspase-9 proteins.

[0065] Figure 8: A. Co-localization detection of SLC44A1 and mitochondrial internal reference protein VDAC1; B. Co-localization detection of SLC44A1 and endoplasmic reticulum stress-related protein calnexin (blue fluorescence: DAPI; red fluorescence: SLC44A1; green fluorescence: VDAC1 and Calnexin).

[0066] Figure 9: A. Expression levels of SLC44A1 in the nucleus and cytoplasm; B. Expression levels of SLC44A1 in mitochondria and cytoplasm excluding mitochondria.

[0067] Figure 10: Protein levels of Bax and cytochrome c in mitochondria and cytoplasm excluding mitochondria.

[0068] Figure 11: Wild-type mice (WT) and SLC44A1 - / - CFU in mouse lungs (n = 3). Detailed Implementation

[0069] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0070] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0071] the term:

[0072] As used in this article, the term "sample" or "sample" refers to material specifically associated with the person being tested, from which specific information relating to the person being tested can be determined or inferred. A sample may consist wholly or partially of biological material from the person being tested.

[0073] PBMCs (peripheral blood mononuclear cells) are the main cell types found in blood that possess a single nucleus. They primarily include lymphocytes (T / B), monocytes, phagocytes, dendritic cells, and a smaller number of other cell types. Lymphocytes constitute a large proportion. The main purpose of isolating PBMCs is to remove multinucleated cells and erythrocytes, thereby easily mimicking the in vitro blood immune environment.

[0074] Ficoll separation: Ficoll is a polymer of sucrose, neutral in nature, with an average molecular weight of 400,000. At a density of 1.2 g / mL, it does not exceed the normal physiological osmotic pressure and does not cross biological membranes. Erythrocytes and granulocytes, being denser, sink to the bottom of the tube after centrifugation; lymphocytes and monocytes, with a density less than or equal to that of the separation medium, float on the surface of the separation medium after centrifugation, although a small number of cells may remain suspended. By aspirating the cells from the surface of the separation medium, mononuclear cells can be separated from peripheral blood. Ficoll is a separation medium used to separate cells of specific densities (three types: 1.077, 1.084, and 1.073 g / mL), suitable for separating cells of known densities.

[0075] Target gene: also known as the target gene, is a structural gene that encodes a protein. Common methods for obtaining target genes include: obtaining them from gene libraries, amplifying them using PCR technology, and artificial synthesis.

[0076] Macrophages: Macrophages are believed to develop from hematopoietic stem cells in the bone marrow, which are then converted into monocytes. After differentiation, they enter the peripheral blood and become circulating monocytes, of which two types have been identified: "inflammatory" and "resident" monocytes, primarily based on the time they remain in the blood before migrating to tissues. Once in tissues, they differentiate into tissue-specific macrophages: including those in the skeletal system (osteoclasts), central nervous system (microglia), lungs (alveolar macrophages), liver (Kupffer cells), and connective tissue (histocytes), as well as those in the spleen, gastrointestinal tract, and peritoneum.

[0077] H37Rv: The first standard strain of Mycobacterium tuberculosis (MTB) was introduced in 1998. The full-length genome of the H37Rv standard strain is approximately 4 million bases long, containing 3,906 protein-coding genes that encode various enzymes involved in lipid metabolism, as well as two glycine-rich protein families, PE and PPE, with repetitive structures. The latter two are what distinguish MTB from other bacteria.

[0078] Example 1: Quantitative RT-qPCR verification of changes in the expression levels of SLC44A1 in monocytes from tuberculosis patients and healthy donors, as well as in H37Rv-infected THP-1 macrophage cell lines and human primary macrophages.

[0079] Experimental methods: Peripheral blood mononuclear cells (PBMCs) from patients with active tuberculosis, latent tuberculosis, and healthy individuals were isolated using Ficoll lymphocyte separation medium density gradient centrifugation. CD14 cells were further obtained using magnetic bead sorting. + The expression of solute carrier family 44member 1 (SLC44A1) in monocytes was detected using qPCR. Similarly, the expression of SLC44A1 was also detected in H37Rv-infected THP-1 macrophages and human primary macrophages. The specific experimental procedures are as follows:

[0080] Ficoll separates PBMCs

[0081] Peripheral blood mononuclear cells (PBMCs) were negatively sorted using magnetic beads to obtain CD14+ monocytes. Peripheral blood was collected from subjects in heparin-anticoagulated tubes and diluted with an equal volume of RPMI 1640 medium. A certain volume of TBD human whole blood mononuclear cell separation medium was pre-added to a 15ml centrifuge tube, and the diluted peripheral blood was slowly added along the tube wall (separation medium: diluted whole blood = 3:4). The tube was then transferred to a horizontal centrifuge at 1000g for 16 minutes with an acceleration of 9 and a deceleration of 0. After centrifugation, the intermediate white membrane layer cells (monocytes) were transferred to a new 15ml centrifuge tube, resuspended in RPMI 1640, and washed twice at 400g for 5 minutes. The cells were then resuspended in RPMI 1640 with 10% fetal bovine serum complete medium and cell counting was performed.

[0082] Stimulation of monocytes in PBMCs to activate into macrophages

[0083] Before the experiment, dissolve colony-stimulating factor (GM-CSF). Before opening the reagent, centrifuge the solution to ensure the contents reach the bottom. After Ficoll separation of PBMCs, count the cells and seed them in 12-well plates at a density of 2 × 10⁶ cells / well. 6 Cells were seeded per well and cultured in complete medium supplemented with GM-CSF (GM-CSF:complete medium = 1:1000, final concentration 25 ng / ml) for 7 days at 37°C in a 5% CO2 incubator. The medium was changed every two days. During medium change, 400g of medium was aspirated from each well and centrifuged for 5 min. The supernatant was discarded, and the cells were resuspended in complete medium containing GM-CSF and added back to the original 12-well plates. Continuous stimulation of PBMCs activated macrophages, which then adhered to the 12-well plates. After washing away the suspended cells with RPMI 1640 medium, cell infection was performed.

[0084] THP-1 cell resuscitation, culture and induction

[0085] The THP-1 and 293T cell lines used in this study were purchased from the Cell Bank of the Chinese Academy of Medical Sciences. Before the experiment, phorbol 12-myristate 13-acetate (PMA) working solution was prepared, and the PMA powder was centrifuged at high speed. Then, it was dissolved in 1 ml of DMSO (concentration 1 mg / ml) and aliquoted. THP-1 cells frozen in liquid nitrogen were quickly transferred to a 37°C water bath until the cryovials thawed. The cells were washed with 5 ml of RPMI medium, centrifuged at 200g for 2 min, the supernatant was discarded, and 10 ml of complete culture medium was added before transferring to cell culture flasks. The medium was changed every two days. Once the cells reached the logarithmic growth phase, they were cultured in 24-well plates at a density of 5 × 10⁶ cells / well. 6 / well, 12-well plate culture density 1×10⁶ / well, 6-well plate culture density 2×10⁶ / well 6 Cells were seeded at a density of 1×10⁶ cells per well and cell culture dish.

[0086] Induction of THP-1 cells using PMA

[0087] Prepare a complete culture medium containing PMA (PMA:complete culture medium = 1:10000, final concentration 100 ng / ml). Add the prepared complete culture medium containing 0.5% PMA to each of 24-well plates, 1 ml to each of 12-well plates, 2 ml to each of 6-well plates, and 10 ml to each cell culture dish. Induce differentiation for 36 hours, and when THP-1 cells have adhered to more than 90% of the culture, stop the induction of cell differentiation and replace the medium with complete culture medium without PMA for another 12 hours of recovery culture. Then, perform cell infection.

[0088] H37Rv recovery and expanded cultivation

[0089] The M. tb standard strain H37Rv was obtained from the Beijing Major Disease Clinical Data and Sample Resource Bank - Tuberculosis Bank. Frozen bacteria were streaked in Middlebrook 7H10 medium at -80℃ and incubated statically at 37℃ for approximately 3 weeks to revive the H37Rv strain. Single colonies from Middlebrook 7H10 medium were inoculated into Middlebrook 7H9 medium and incubated at 37℃ for approximately 2 weeks until the optical density (A) reached a certain value. 600 When the value reaches 0.6-0.8, it indicates that H37Rv has grown to the logarithmic growth phase and is used for infection. Aseptic technique is maintained throughout the process.

[0090] Construction of cell infection model

[0091] After inducing cell adhesion, the cells were infected with H37Rv strain. One ml of bacteria in logarithmic growth phase and one ml of sterile culture medium (7H9 + 10% OADC) were placed in a cuvette for OD measurement (1 OD = 3 × 10⁻⁶). 8Cells were infected with a multiplicity of infection (MOI) of 10. The required bacterial load was calculated and transferred to 15 ml centrifuge tubes. An appropriate amount of RPMI 1640 medium was added, and the cells were centrifuged at 4500 rpm for 7 min at room temperature for washing. After centrifugation, the supernatant was discarded, and the cells were resuspended in 1 ml of complete culture medium. The suspension was then transferred to dispersion tubes and sonicated. Finally, the sonicated bacterial suspension was added to a fixed volume of complete culture medium, mixed thoroughly, and then transferred to individual wells of a plate. The plates were incubated at 37°C with 5% CO2. After 4 h, the culture medium in the wells was aspirated, and the cells were washed three times with RPMI 1640 medium to remove extracellular M. tb. Cells, RNA, proteins, or intracellular bacteria were collected at different time points as needed for corresponding cell phenotype and CFU analysis.

[0092] RNA extraction

[0093] Total RNA was extracted from the samples using the miRNeasy Mini Kit (217004, Qiagen). 30 ml of anhydrous ethanol (analytical grade) was added to Wash Buffer 1 (RWT) and mixed by inverting. 44 ml of anhydrous ethanol (analytical grade) was added to Wash Buffer 2 (RPE) and mixed. The samples lysed with 700 μl of QIAzol were removed from the -80°C freezer and allowed to thaw for 5 min. 140 μl of chloroform was added, and the mixture was thoroughly vortexed and allowed to stand for 2-3 min. Then, the mixture was centrifuged at 12000 g for 15 min at 4°C. After centrifugation, the upper clear aqueous phase was transferred to a new enzyme-free centrifuge tube. 1.5 volumes of anhydrous ethanol were added, and the mixture was mixed by pipetting. The mixture was then transferred to a collection column, centrifuged at 10000 g for 15 s, the filtrate was discarded, and 700 μl of Wash Buffer 1 was added. The mixture was then centrifuged at 10000 g for 15 s. Discard the filtrate, add 80 μl of DNase (52 μl enzyme-free water, 20 μl DNase, 8 μl reaction buffer), and incubate at room temperature for 15 min. Add 700 μl of wash buffer 1 and 500 μl of wash buffer 2 in fractions, centrifuge at 10000g for 15 s at room temperature, and discard the filtrate. Add 500 μl of wash buffer 2, centrifuge at 10000g for 1 min, transfer the collection column to a new centrifuge tube, open the cap, and centrifuge at 20000g for 1 min at room temperature. Transfer the collection column to the centrifuge tube, add 30 μl of RNase-free ddH2O, incubate for 2 min, centrifuge at 10000g for 1 min, and recover the liquid in the centrifuge tube into the collection column. Repeat centrifugation once to increase the total RNA yield. Determine the RNA concentration and purity using a NanoDrop2000 ultraviolet spectrophotometer.

[0094] 8. mRNA reverse transcription and qPCR, the specific methods are as follows:

[0095] 1) The reagent kit used for mRNA reverse transcription was the ReverTra Ace qPCR RT Kit (TOYOBO, Japan). The mRNA reverse transcription system is shown in Table 1.

[0096] Table 1. mRNA reverse transcription system (dye method)

[0097]

[0098] 2) The conditions for reverse transcription are: .

[0099] 3) GAPDH is the internal reference gene in mRNA detection; the kit used for qPCR detection is PowerUp SYBR GreenMaster Mix (Applied Biosystems, USA), and the PCR reaction system is shown in Table 2.

[0100] Table 2 qPCR reaction system (dye method)

[0101]

[0102] 4) The PCR reaction program is set as follows:

[0103]

[0104] After the PCR reaction was completed, the experimental data were exported and analyzed using the relative expression level method (2). -ΔΔCt The expression levels of mRNA were analyzed.

[0105] 9. Primers used in this study are shown in Table 3.

[0106] Table 3 Primers and their sequences used in this study

[0107]

[0108] (B) Results: The expression level of SLC44A1 in monocytes of patients with active tuberculosis was significantly higher than that in the control group. At the THP-1 cell level, the expression level of SLC44A1 was significantly increased 24 h and 48 h after H37Rv infection compared with the uninfected group (P<0.01); at the human primary macrophage level, the expression level of SLC44A1 in cells after H37Rv infection was significantly increased compared with the control group (P<0.001). In isolated mouse peritoneal primary macrophages, the expression level of SLC44A1 in the H37Rv-infected group was significantly higher than that in the control group (P<0.01); similarly, the expression level of SLC44A1 in mouse lung tissue was significantly higher than that in the control group (P<0.05). These results indicate that H37Rv infection leads to the upregulation of SLC44A1 expression.

[0109] Example 2: SLC44A1 protein's resistance to mycobacterial infection

[0110] Experimental Methods: Three SLC44A1-specific silence RNAs (siRNAs) provided by Guangzhou Ruibo Biotechnology Co., Ltd., and their control siRNA-NC, were used for preliminary transfection efficiency testing. siRNA-SLC44A1-02 was ultimately selected for subsequent functional studies of SLC44A1. SLC44A1-low expression THP-1 cells and normal THP-1 cells were infected with H37Rv, and intracellular CFU at different time points was measured to demonstrate the functional role of SLC44A1 in anti-pathogen processes. For example, in transfecting 12-well plates, the siRNA transfection concentration was 50 nM, and the transfection reagent ratio was Lipofectamine™ RNAiMAX: siRNA = 5:1.

[0111] The specific transfection steps are as follows:

[0112] Add 150 μl of Opti-MEM® culture medium to tube A, then add 12.5 μl of Lipofectamine™ RNAiMAX and gently pipette to mix.

[0113] Add 150 μl of Opti-MEM® medium to tube B, then add 2.5 μl of siRNA and mix well by pipetting.

[0114] Add the liquid from tube B to tube A, mix well, and let stand for 15 minutes.

[0115] Remove the plated THP-1 cells from the cell culture incubator, wash away the PMA, discard the original culture medium, and add 700 μl of complete culture medium.

[0116] Add the liquid from tubes A and B to 700 μl of pre-added complete culture medium and mix well. Return the 12-well plate to the incubator and continue culturing.

[0117] Results: Comparing the differences in CFU in cells between the normal THP-1 cell group (NC siRNA) and the SLC44A1-low expression THP-1 cell group (SLC44A1 siRNA group), the survival rate of H37Rv in the SLC44A1 siRNA group was significantly higher than that in the NC siRNA group after 24 h and 48 h of infection. The results indicate that the clearance rate of mycobacteria in THP-1 cells with normal SLC44A1 expression is faster, while the survival rate of mycobacteria is enhanced in THP-1 cells with low SLC44A1 expression.

[0118] Example 3: SLC44A1 protein regulates apoptosis and inhibits H37Rv survival in cells.

[0119] Experimental Methods: SLC44A1-specific siRNA and control siRNA were transfected into THP-1 cells to construct SLC44A1-silenced cells and their control cells. SLC44A1-silenced THP-1 cells and normal THP-1 cells were infected with H37Rv, respectively. After Annexin V / 7-AAD staining, the results were analyzed by flow cytometry. Protein samples were collected from H37Rv-infected and uninfected SLC44A1-silenced cells and their control cells. After BCA concentration determination, the expression of caspase-3 and cleaved caspase-3 was detected by Western blot.

[0120] The steps for Western blot detection of protein expression levels are as follows:

[0121] Cell protein collection and extraction

[0122] THP-1 cells were induced in 6-well plates at a cell density of 2 × 10⁻⁶ cells / well. 6 / well. After PMA induction for 36 h, the medium was replaced with complete medium without PMA, and the cells were cultured for 12 h. H37Rv was used to infect cells with an MOI of 10, and cell samples were collected from uninfected cells, cells infected for 24 h, and cells infected for 48 h. Clean the orifice plate once, add Gently scrape off the cells, mix them by pipetting, and then transfer the liquid to a 1.5ml centrifuge tube. Centrifuge at 400g for 5 minutes, discard the supernatant, and freeze the cell pellet at -80℃ for later use.

[0123] Prepare protein lysis buffer before protein extraction: Add 100 μl of phosphatase inhibitor and 10 μl of protease inhibitor to 890 μl of RIPA lysis buffer. Remove protein samples from the -80°C freezer and place them on ice. Add 60 μl of protein lysis buffer to each sample, mix well by pipetting, and lyse on ice for 20 min. Then centrifuge at 20,000 g for 20 min at 4°C. Transfer the supernatant (60 μl) to a new 1.5 ml centrifuge tube, mix well, and centrifuge for later use (store samples at low temperature throughout the process).

[0124] BCA method for determining protein concentration

[0125] Add 1.2 ml of protein standard preparation solution to one tube of protein standard (30 mg BSA), dissolve thoroughly to prepare a 25 mg / ml protein standard solution. Dilute the 25 mg / ml protein standard solution to a final concentration of 0.5 mg / ml using RIPA lysis buffer. Then aliquot into 70 μl tubes. Simultaneously, prepare BCA working solution before the experiment (prepare fresh). Based on the number of samples (200 μl per sample), prepare an appropriate amount of BCA working solution at a ratio of 50 volumes of BCA reagent A to 1 volume of BCA reagent B (50:1), mix thoroughly, and set aside.

[0126] Add 0.5 mg / ml standard to the standard wells of a 96-well plate at concentrations of 0, 1, 2, 4, 8, 12, 16, and 20 μl, respectively. Then, add standard dilution buffer (RIPA lysis buffer) to bring the total volume to 20 μl, corresponding to standard concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / ml. Pipette 4 μl of sample into 16 μl of RIPA lysis buffer (sample diluted 5-fold), mix, centrifuge, and transfer to the sample wells of the 96-well plate. Then, using a multi-channel pipette, add 200 μl of BCA working solution to both the standard and sample wells simultaneously. Incubate at 37°C for 20-30 min. The color in the standard wells will deepen with increasing standard concentration. Measure the absorbance between 540-595 nm (550 nm) using a microplate reader. Then, calculate the protein concentration of the sample based on the standard curve and the sample volume used. After the protein concentration was measured, 11.2 μl of 6× loading buffer was added to each sample tube and boiled in a metal bath at 95°C for 5 min to denature the protein and prevent its degradation.

[0127] 3. SDS-PAGE polyacrylamide gel electrophoresis

[0128] (1) Fix the glass plates horizontally, prepare the separating gel (formula in Table 4), inject 8 ml of separating gel between the glass plates, add anhydrous ethanol to press the lines, and after the separating gel solidifies, pour off the anhydrous ethanol and let the remaining anhydrous ethanol evaporate naturally; then prepare the stacking gel (formula in Table 4), fill the space between the glass plates with stacking gel and insert the corresponding comb, and let it stand at room temperature for 30 min. After the gel solidifies, carefully remove the comb between the glass plates, put the glass plates into the electrophoresis tank, fill the inside with electrophoresis buffer, and rinse the sample wells with electrophoresis buffer to avoid air bubbles in the wells, and fill the outside with 1 / 2 electrophoresis buffer; load 10 μg of protein and 5 μl of marker; use 80V for the upper gel, and adjust the voltage to 120V when the sample migrates to the separating gel, and continue electrophoresis until the sample migrates to the bottom of the separating gel.

[0129] Table 4. Formulations for separating gels and stacking gels used in protein electrophoresis.

[0130]

[0131] (2) After electrophoresis, peel the gel off the glass plate and cut off any excess gel. Soak a stack (7 sheets) of filter paper completely in the transfer buffer. Activate the nitrocellulose membrane (PVDF membrane) in methanol for about 15 seconds, then equilibrate it in the transfer buffer for 1 minute. Place it on the soaked filter paper, then place the gel on the PVDF membrane. Finally, cover with another stack of filter paper, press the plate to remove air bubbles from the gaps, place it in the transfer tank, and tighten the safety cap. Transfer one gel at 1.3A current and 25V voltage for 20-30 minutes; transfer two gels together at 2.5A current and 25V voltage for 20-30 minutes. After transfer, remove the PVDF membrane and cut it according to the target protein, keeping the cut blank membrane (for subsequent whole-membrane imaging). Place the PVDF membrane in blocking buffer (use 5% BSA or 5% skim milk according to the antibody instructions), block at room temperature for 1 hour. After blocking, discard the blocking buffer, add the primary antibody diluted in the blocking buffer, and incubate overnight at 4°C with shaker (14-16 hours). After primary antibody incubation, recover the antibody and add PBST (…). Wash four times with 10% Tween 20 solution, 10 min each time. Add HRP-labeled secondary antibody diluted with blocking buffer and incubate on a shaker for 1 h. After secondary antibody incubation, discard the antibody and wash four times with PBST, 7 min each time. After washing, prepare developing solution (solution A:solution B = 1:1), apply the developing solution to the PVDF membrane, and expose the protein bands using a chemiluminescence analyzer. Use ImageJ to calculate the protein grayscale value to determine the relative protein expression level.

[0132] The steps for analyzing cell apoptosis using flow cytometry are as follows:

[0133] Apoptosis was detected using Annexin V combined with 7-AAD double staining. THP-1 cells were induced in 12-well plates and subsequently infected with H37Rv. Cell samples were collected at 0h, 24h, and 48h for apoptosis detection. Wash the cells in the wells of the plate, add 400 μl of 0.25% trypsin, and incubate at 37°C for 5 min to digest the cells. Then, add an equal volume of complete culture medium to stop the digestion reaction. Transfer the liquid from the wells to a centrifuge tube, centrifuge at 400g for 5 min, discard the supernatant, and add... Rinse the cells, centrifuge at 400g for 5 minutes, and discard the supernatant; Resuspend the supernatant and add 5 μl of PE or APC-labeled Annexin V and 5 μl of 7-AAD. Incubate at room temperature in the dark for 30 min. Rinse the cells twice, finally Cells were resuspended and apoptosis was detected using flow cytometry (FACS).

[0134] Results: At 24 and 48 hours after infection, the survival rate of H37Rv in cells infected with SLC44A1 siRNA was significantly higher than that in the NC siRNA group, indicating that SLC44A1 can inhibit the survival of H37Rv in macrophages. At 24 and 48 hours after H37Rv infection, the expression level of cleaved caspase-3 protein in the SLC44A1 siRNA group was significantly lower than that in the control group. These results suggest that SLC44A1 has the function of promoting macrophage apoptosis.

[0135] Example 4: SLC44A1 protein inhibits H37Rv survival in cells by regulating endogenous apoptosis.

[0136] Experimental methods: SLC44A1 siRNA group and control group cells were infected with H37Rv, and the expression of caspase-8, cleaved caspase-8, caspase-9, and cleaved caspase-9 in SLC44A1 siRNA group and control group after H37Rv infection was detected.

[0137] Results: 24 h and 48 h after H37Rv infection, the expression level of cleaved caspase-9 in SLC44A1 silenced cells was significantly lower than that in the uninfected group, while the expression level of cleaved caspase-8 after H37Rv infection did not change significantly compared with the uninfected group. These results suggest that SLC44A1 may regulate macrophage apoptosis through the caspase-9-mediated intrinsic apoptosis pathway.

[0138] Example 5: SLC44A1 inhibits H37Rv survival in cells via mitochondrial-mediated endogenous apoptosis.

[0139] Experimental Methods: Confocal microscopy was used to detect the co-localization of SLC44A1 with mitochondrial and endoplasmic reticulum proteins. Subcellular components of THP-1 cells were isolated, and Western blot was used to detect the intracellular localization of SLC44A1 protein. SLC44A1-silenced cells and control cells were infected with H37Rv, respectively, to test the translocation of mitochondrial apoptosis-related proteins Bax and cytochrome c from mitochondria to the cytoplasm.

[0140] The specific steps for immunofluorescence staining and confocal analysis of the intracellular localization of SLC44A1 are as follows:

[0141] THP-1 cells were induced in small glass dishes (containing glass slides) at a cell density of 4 × 10⁻⁶ cells / mL. 6 / plate. After induction for 36 hours, replace with complete medium without PMA and allow to recover for 12 hours. Wash cells for 5 minutes, add 1 ml of 4% paraformaldehyde, and incubate at room temperature for 10 minutes to fix the cells. Cells were washed three times, 5 minutes each time; next, the cells were permeabilized to detect intracellular target proteins using a solution containing 0.2% Triton X-100. Incubate cells for 5 minutes. Cells were washed three times, 5 minutes each time; then a solution containing 1% BSA, 1‰ Tween 20, and 22.52 mg / ml glycine was applied. Incubate cells for 30 minutes to block non-specific antibody binding. Use a solution containing 1% BSA and 1‰ Tween 20. Dilute the antibodies (SLC44A1 antibody concentration 1:50; VDAC1 antibody concentration 1:100; Calnexin antibody concentration 1:100) and add them to the corresponding glass dishes (SLC44A1 is co-incubated with VDAC1 and Calnexin respectively), incubate overnight at 4°C, then... Wash cells three times, 5 minutes each time; then dilute the fluorescent antibody (secondary antibody 1:1000; goat anti-rabbit is red fluorescent, goat anti-mouse is green fluorescent), and incubate in the dark for 1 hour. Wash cells three times in the dark for 5 minutes each time; label cell nuclei with DAPI (DAPI: 1×PBS = 1:99), incubate in the dark for 5 minutes, then wash cells three times in the dark with 1×PBS for 5 minutes each time; finally, block cell slides with mounting medium (mounting medium: glycerol = 1:50) and store at -20℃ or 4℃ in the dark.

[0142] Fluorescence was captured using a laser confocal scanning microscope. Cell fields were observed under a 60x objective lens. Cy3 (red fluorescence), FITC (green fluorescence), DAPI (blue fluorescence), and brightfield were selected from the dye list. Parameters such as detector sensitivity (HV) for each color fluorescence were adjusted. Finally, the spectrum was scanned and photographed to obtain target images. ImageJ was then used to perform colocalization analysis on the image results.

[0143] The steps for separating cell subcomponents are as follows:

[0144] Separating the cell nucleus and cytoplasm to detect the intracellular localization of SLC44A1

[0145] With 2×10 6 Cells were seeded at a density of / well in 6-well plates. After PMA induction for 36 h, H37Rv infection was performed. Cell components were separated using a nucleocytoplasmic separation kit 24 h post-infection. 400 μl of trypsin was added to each well, and the plate was incubated at 37°C for digestion. Complete culture medium was added after 5 min to terminate the trypsin digestion. Cells were collected by centrifugation at 200 g for 5 min. The cells were pre-cooled. After washing the cells, centrifuge at 200g for 5 min, discard the supernatant, add 300 μl of pre-chilled fractionation buffer, gently mix, and incubate on ice for 7 min. Then centrifuge at 500g for 5 min at 4°C, until a precipitate is observed; the precipitate is the total nuclear protein, and the aspirated supernatant is the total cytoplasmic protein. Lyse the nuclear proteins using RIPA lysis buffer, and determine the protein concentration using BCA for subsequent protein detection.

[0146] Isolate cellular mitochondria to detect mitochondrial apoptosis-related proteins

[0147] All solutions in the room temperature thaw kit (mitochondrial isolation reagent, mitochondrial lysis buffer, and protease inhibitor PMSF) should be thawed, placed on ice immediately after thawing, and mixed well. Then, aliquot the solutions into 15ml and 1.5ml centrifuge tubes.

[0148] With 1×10 7 Cell density in each well: THP-1 cells were seeded in 10 cm diameter cell culture dishes and induced with PMA for 36 h, followed by inhibition of SLC44A1 expression using siRNA. Mitochondrial cells were isolated 24 h after H37Rv infection. First, [the following was applied]... Wash the cells, add 3 ml of trypsin cell digestion solution evenly, and incubate at 37°C for digestion. After 5 minutes, add an equal volume of complete culture medium to terminate the trypsin digestion. Centrifuge at 400g at room temperature for 5 minutes to collect the cells. Use pre-cooled... Cells were washed and centrifuged at 4°C for 5 min to precipitate the cells. The cells were then resuspended in 1 ml of mitochondrial separation reagent and incubated on ice for 10-15 min. The cell suspension was transferred to a glass homogenizer, homogenized, and centrifuged at 600 g for 10 min at 4°C. The supernatant was transferred to another centrifuge tube and centrifuged at 11000 g for 10 min at 4°C. The supernatant was then transferred to a new centrifuge tube, and the collected pellet was the isolated mitochondria. This pellet was lysed using 100 μl of mitochondrial lysis buffer. The supernatant was then centrifuged at 12000 rpm for 10 min at 4°C, and the collected supernatant was the cytoplasmic protein with mitochondria removed. The protein concentrations of mitochondrial proteins and mitochondrial-removed cytoplasmic proteins were determined using the BCA method. Finally, Western blot analysis was performed to detect changes in protein expression.

[0149] Results: Colocalization analysis revealed that the fluorescent spots of SLC44A1 overlapped with those of the mitochondrial internal reference protein VDAC1, but not with those of the endoplasmic reticulum stress-related protein calnexin. Western blot analysis showed that SLC44A1 was mainly located in the cytoplasmic mitochondria. During mitochondrial-mediated endogenous apoptosis, SLC44A1 inhibited Bax entry into mitochondria and simultaneously inhibited cytochrome c translocation from mitochondria to the cytoplasm, resulting in a low mitochondrial apoptosis-inducing state. These results indicate that SLC44A1 controls intracellular Mycobacterium tuberculosis survival by regulating the mitochondrial-mediated endogenous apoptosis pathway.

[0150] Example 6: In vivo validation of SLC44A1 promoting host resistance to Mycobacterium tuberculosis infection in mice (A) Experimental methods: Specific pathogen-free (SPF) grade female C57BL / 6J mice, 4-6 weeks old, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. SLC44A1 - / - Mice were provided by Guangzhou Cyagen Biotech Co., Ltd. Mice were housed in a temperature-controlled negative pressure animal room, with 6 mice per cage, where food and water were readily available. Mouse experiments were conducted after approval by the Animal Ethics Committee of Beijing Chest Hospital. Wild-type mice and SLC44A1 mice were infected with H37Rv. - / - Mice were used, and lung tissue was collected 14 days later for CFU and pathological examination.

[0151] The steps to establish a mouse model of tuberculosis infection are as follows:

[0152] Dilute H37Rv (10) with sterile PBS + 0.05% Tween-80. 7(CFU / ml), sonicated for 1 min, and the final bacterial suspension was brought to a final volume of 5 ml. Mice to be infected were placed in baskets, the chamber was closed, and the bacterial suspension was aspirated into the nebulizer. Nebulization infection and sterilization were performed following a 15-min preheating, 30-min nebulization, 30-min smoke decay, and 15-min purification cycle. After infection, mice were housed separately in a negative pressure infection animal room. On the first day after infection, three mice were dissected to count CFU and determine the success of the model.

[0153] (B) Results: The degree of pathological inflammatory damage in wild-type mice was significantly lower than that in SLC44A1. - / - Mice. The results showed that SLC44A1 could inhibit the survival of Mycobacterium tuberculosis in macrophages and mouse lung tissue, that is, SLC44A1 has the effect of inhibiting the proliferation of M. tuberculosis in vivo.

Claims

1. The use of SLC44A1 in the preparation of drugs for treating Mycobacterium tuberculosis infection, wherein the amino acid sequence of SLC44A1 is shown in SEQ ID NO.

1.

2. The application as described in claim 1, wherein the Mycobacterium tuberculosis infection includes pulmonary tuberculosis infection, lymphatic tissue tuberculosis infection, and bone and joint tuberculosis infection.

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