Use of hif-2α and related substances in the treatment of mycobacterial infections

Abstract

The application relates to the field of biological medicine, and provides application of HIF-2alpha and related substances in anti-mycobacterium infection. Specifically relates to the use of HIF-2alpha protein and a nucleic acid molecule encoding the protein in the preparation of products for preventing and / or treating mycobacterium tuberculosis and related non-tuberculous mycobacterium (NTM) infection diseases. HIF-2alpha can inhibit intracellular proliferation of mycobacterium, reduce inflammation and damage of tissues, provide a new target for the treatment of mycobacterium infection, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of acetyltransferase HIF-2α and related substances in the preparation of drugs against mycobacterial infections. Background Technology

[0002] Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb). The World Health Organization estimates that approximately 10 million new TB cases are diagnosed globally each year, and up to 1.5 million people die from it. The main reasons for the severe TB epidemic include: 1) a single vaccine with limited preventative effect; 2) the prevalence of drug-resistant TB and low treatment success rates; and 3) complex treatment regimens and long treatment courses. Solving these problems requires a deeper understanding of the interaction mechanisms between Mtb and the host immune system, providing a theoretical basis for finding new treatment strategies.

[0003] The emergence and development of host-directed therapy (HDT) can serve as adjunctive therapy for infections caused by multidrug-resistant strains. Its mechanism involves intervening in the expression and function of host genes, enhancing the host's protective immune response, increasing its ability to kill Mycobacterium tuberculosis, and reducing the inflammatory response to decrease inflammation and pathological damage caused by bacterial infection, thereby synergistically achieving better therapeutic effects with anti-tuberculosis drugs. Unlike anti-tuberculosis drugs that directly target Mycobacterium tuberculosis, HDT has the following advantages: First, it is effective against both susceptible and drug-resistant Mycobacterium tuberculosis; second, it carries a lower risk of inducing drug resistance in Mycobacterium tuberculosis; and third, it can shorten the duration of antibiotic treatment by targeting multiple components and signaling pathways of the host's immune system.

[0004] In summary, there is an urgent need in this field to develop an active substance that can effectively block mycobacterial invasion, effectively resist mycobacterial infection, and control tissue inflammatory damage caused by mycobacterial infection, in order to control mycobacterial infectious diseases and their related symptoms and / or other complications.

[0005] Hypoxia-inducible factor HIF-2α participates in hypoxia response, iron homeostasis and inflammation regulation in various tumors, hypoxia adaptation, cardiovascular and infectious diseases, and affects monocyte polarization and antibacterial function, but its expression profile and function in immune cells of tuberculosis patients are still unclear. Summary of the Invention

[0006] This invention is based on the above research and studies the application of hypoxia-inducible factor HIF-2α and its related substances in the preparation of drugs against mycobacterial infections.

[0007] Multi-omics technologies can simultaneously analyze two layers of information: expression and regulation. RNA-seq quantifies transcripts, reflecting the activation status of functional pathways; ATAC-seq depicts chromatin accessibility and locates regulatory elements such as transcription factors. The combination of these two technologies can link the activation of regulatory elements with target gene expression and pathway changes, characterizing immune remodeling at the mechanistic level. This study used peripheral blood mononuclear cells from tuberculosis patients to construct a combined transcriptome and chromatin accessibility map, systematically evaluating the expression characteristics and potential functions of HIF-2α, providing a basis for HDT target discovery.

[0008] This study included treatment-naïve drug-sensitive tuberculosis patients (n=17) and healthy controls (n=29). Peripheral blood was collected and mononuclear cells were obtained using magnetic bead sorting. RNA-seq and ATAC-seq were performed. Differentially expressed genes (DEGs) and chromosome accessibility (DARs) analyses, combined with pathway enrichment and transcription factor binding site characteristics, were used to elucidate the epigenetic regulatory remodeling of immune and metabolic pathways. The effects of candidate factors on macrophage antibacterial function were then validated in a Mycobacterium marinum-macrophage infection model.

[0009] The results showed that the gene encoding hypoxia-inducible factor HIF-2α, EPAS1, was significantly overexpressed in peripheral blood immune cells of patients with active tuberculosis; inhibition of HIF-2α in macrophages significantly increased the proliferation of Mycobacterium tuberculosis in macrophages; and promotion of HIF-2α expression significantly inhibited the proliferation of Mycobacterium tuberculosis.

[0010] Based on the above research, the technical solution to be protected by this invention is as follows:

[0011] One of the main objectives of this invention is to provide the use of HIF-2α in combating mycobacterial infections, and further to provide its use in treating mycobacterial infectious diseases and related diseases or symptoms. The medicines or pharmaceutical compositions of this invention can be used to effectively combat mycobacterial infections and control the occurrence of infectious diseases.

[0012] In a first aspect disclosed in this invention, the use of an HIF-2α promoter in the preparation of products for the prevention and / or treatment of mycobacterial infectious diseases and / or infection-related diseases and / or symptoms is provided.

[0013] Preferably, the HIF-2α promoter is selected from any one or more of the following: (1) exogenous HIF-2α protein or the nucleic acid EPAS1 encoding it; (2) liposomes, nanomaterials, PEG-modified proteins, protein microspheres or recombinant expression vectors that encapsulate the nucleic acid encoding HIF-2α.

[0014] In a second aspect disclosed in this invention, a product is provided with HIF-2α or a nucleic acid molecule encoding the protein as an active component.

[0015] In a third aspect disclosed in this invention, a method is provided for treating mycobacterial infectious diseases and / or alleviating infection-related diseases and / or symptoms, the method comprising administering a therapeutically effective amount of HIF-2α, a nucleic acid molecule encoding the protein, or a recombinant expression vector thereof to a subject in need.

[0016] In a fourth aspect disclosed in this invention, the application of HIF-2α in the preparation of mycobacterial detection kits or antimicrobial drug screening kits is provided for the detection of mycobacteria or the screening of antimycobacterial drugs.

[0017] Preferably, the mycobacteria are selected from one or more of the following group: Mycobacterium tuberculosis, Mycobacterium marineum, avian-intracellular mycobacterial complex, Mycobacterium kansas, Mycobacterium simianum, Mycobacterium bufotatum, Mycobacterium Gordonum, Mycobacterium terrestrialum, and Mycobacterium paraterrestrialum. In a preferred embodiment of the present invention, Mycobacterium marineum is used as the experimental subject.

[0018] The disease or symptoms associated with mycobacterial infection are selected from one or more of the following: histopathological damage caused by mycobacterial infection; insufficient or excessive production of cytokines after infection; inflammatory damage to organs; and multiple organ failure, for example, the organs are selected from: lung, kidney, brain, stomach, and intestine.

[0019] The HIF-2α-encoded nucleic acid is selected from the EPAS1 sequence or fragment, or from the EPAS1 mRNA, cDNA, or precursor.

[0020] In a preferred embodiment of the present invention, the HIF-2α protein is selected from any of the following:

[0021] (a) A polypeptide having the amino acid sequence shown in SEQ ID NO.2;

[0022] (b) Substances that are homologous or identical to the amino acid sequence shown in SEQ ID NO.2 and that promote HIF-2α function;

[0023] Substances that have undergone substitution, deletion, or addition of one or more amino acids in the amino acid sequence of (c), (a), or (b) and have the function of promoting HIF-2α, or proteins or polypeptides derived from (a) or (b),

[0024] The nucleic acid molecule encoding the HIF-2α protein is selected from any of the following:

[0025] (i) A nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO.1;

[0026] (ii) Molecules that hybridize with the nucleotide sequence defined in (i) under strict conditions;

[0027] (iii) Nucleic acid molecules that are homologous to or have the same sequence as the nucleotide sequence shown in SEQ ID NO.1;

[0028] (iv) A nucleic acid molecule consisting of one or more nucleotides substituted, deleted or added from the nucleotide sequence in (i), (ii) or (iii).

[0029] In some embodiments, the HIF-2α protein is: a naturally purified protein, a chemically synthesized product, or produced from a prokaryotic or eukaryotic host using recombinant technology. The host is selected from bacteria, yeast, higher animal and mammalian cells, preferably human HIF-2α.

[0030] Furthermore, the present invention also provides a recombinant HIF-2α expression vector, comprising an expression vector and a nucleic acid molecule encoding the HIF-2α protein inserted onto the expression vector, wherein the nucleic acid molecule encoding the HIF-2α protein is as described above.

[0031] The expression vector is a conventional vector such as a plasmid vector, granular vector, phage vector, or viral vector, and the specific type is selected from existing technologies based on the actual situation. The "viral vector" includes adeno-associated virus and lentivirus. Suitable viral vectors are well known to those skilled in the art. Other "non-viral vectors" include liposomes or lipid complexes, cationic polymers, chitosan polymers, and nanoparticle carriers. Suitable non-viral vectors are well known to those skilled in the art.

[0032] In the second and third aspects disclosed in this invention, an anti-mycobacterial product is provided, said product being a pharmaceutical composition, for example, in a form suitable for administration by means of a pharmaceutical composition or kit selected from the group consisting of: oral administration, injection (e.g., direct naked DNA or protein injection, liposome-encapsulated DNA or protein injection), gold-coated gene gun bombardment, plasmid DNA carried by reproduction-deficient bacteria, target DNA carried by replication-deficient adeno-associated virus, or nasal administration, pulmonary administration, oral administration, or transdermal administration of a protein encoded by a target gene.

[0033] Preferably, the pharmaceutical composition of the present invention comprises:

[0034] (A) Therapeutic or preventative effective amounts of this HIF-2α, the nucleic acid molecule encoding the protein, and / or its promoters;

[0035] (B) Pharmaceutically or immunologically acceptable carriers or excipients;

[0036] (C) Optionally, one or more other active substances for the prevention or treatment of mycobacterial infectious diseases and related symptoms and / or conditions.

[0037] Under normal circumstances, liquid formulations can be stored stably at 2℃-8℃ for at least one year, while lyophilized formulations remain stable at 30℃ for at least six months. The formulations can be commonly used in the pharmaceutical industry, such as suspensions, injections, and lyophilized formulations.

[0038] When the composition of the present invention is administered to animals, including humans, the dosage varies depending on the patient's age and weight, disease characteristics and severity, and route of administration. The dosage can be determined with reference to the results of animal experiments and various other factors, but the total dosage should not exceed a certain range.

[0039] In a fourth aspect, the present invention provides the application of reagents for detecting HIF-2α expression levels in the preparation of mycobacterial detection kits or antimicrobial drug screening kits, and corresponding kit products.

[0040] The experimental results showed that HIF-2α expression was significantly upregulated in patients with mycobacterial infection, suggesting that it is a protective factor of the body's immune response and has the effect of inhibiting the occurrence and development of mycobacteria. Based on this, it can be inferred that HIF-2α can be used as a marker for mycobacterial detection or a marker for screening antimicrobial drugs.

[0041] The reagent used to detect HIF-2α expression level is selected from reagents for detecting HIF-2α protein expression level, or it can be a reagent for detecting EPAS1 gene expression level.

[0042] The method for drug screening using HIF-2α as a biomarker is as follows:

[0043] (A) Treating cells, tissues, or animals infected with mycobacteria or simulating symptoms of infection with the candidate substance;

[0044] (B) Detect the level of HIF-2α protein or the nucleic acid molecule encoding the protein in the cells, tissues or animals;

[0045] (C) If, after treatment with the candidate substance, the level of HIF-2α protein or the nucleic acid molecule encoding the protein is higher than the expression level in the infected model tissues or cells, it indicates that the candidate substance has the effect of treating mycobacterial infection by promoting HIF-2α.

[0046] In a sixth aspect, the present invention provides a mycobacterial detection kit or an antimicrobial drug screening kit, which comprises reagents for detecting the expression level of EPAS1 (the gene encoding HIF-2α) in biological samples and consists of a reverse transcription system, a primer system, and an amplification system. Diagnosis or drug screening is achieved by detecting the nucleic acid expression level of EPAS1.

[0047] The primer sequences with specificity for detecting HIF-2α in biological samples are shown in SEQ ID NO. 3 and 4 below:

[0048] EPAS1-F: 5'-CTGTGTCTGAGAAGAGTAACTTCC-3' (SEQ ID NO.3);

[0049] EPAS1-R: 5'-TTGCCATAGGCTGAGGACTCCT-3' (SEQ ID NO. 4).

[0050] The beneficial protections and effects of this invention are as follows:

[0051] This invention provides the use of HIF-2α in products for treating mycobacterial infectious diseases and / or diseases and / or symptoms related to mycobacterial infection. Experiments have demonstrated that HIF-2α inhibition promotes mycobacterial invasion and infection of cells, suggesting that HIF-2α is a protective factor against mycobacterial infection and has an inhibitory effect on mycobacterial invasion and infection. HIF-2α activation can inhibit mycobacterial infection, manifested by inhibiting the proliferation of mycobacteria in macrophages, thereby achieving the treatment of mycobacterial infectious diseases. Therefore, this invention provides a new target for the treatment of mycobacterial infectious diseases by targeting the functional activation of HIF-2α to inhibit mycobacterial proliferation, and has broad clinical application prospects. Attached Figure Description

[0052] The present disclosure will be further described below with reference to the accompanying drawings, which are shown only for illustrating the embodiments of the present disclosure and are not intended to limit the scope of the present disclosure.

[0053] Figure 1 The study revealed elevated HIF-2α expression levels in tuberculosis-associated monocytes and macrophages: A, RNA-seq data showed increased EPAS1 mRNA expression levels in monocytes of tuberculosis patients compared to healthy controls. B, RT-qPCR validation of EPAS1 expression in an independent clinical cohort also showed significantly higher EPAS1 expression levels in monocytes of tuberculosis patients than in healthy controls. C, In an in vitro infection model, epis1 expression levels were detected at different time points after infection with Mycobacterium tuberculosis. Results showed significantly elevated epis1 expression at both 24 and 48 hours post-infection, indicating that mycobacterial infection can induce upregulation of HIF-2α expression in macrophages. Significance: * P < 0.05; *** P < 0.001.

[0054] Figure 2The study showed increased proliferation of mycobacteria in macrophages after HIF-2α inhibition. AB, immortalized bone marrow-derived macrophages (iBMDM) were transfected with small interfering RNA targeting Epas1 to reduce HIF-2α expression. After transfection, Epas1 mRNA levels significantly decreased, and Western blot results showed a simultaneous decrease in HIF-2α protein levels. C, the cells were infected with Mycobacterium tuberculosis, and the number of bacteria in the cells after infection was detected. The results showed that the bacterial load in macrophages in the Epas1 knockdown group was significantly increased compared to the control group. DE, macrophages were treated with a small molecule inhibitor of HIF-2α to inhibit HIF-2α function. The results showed that after HIF-2α activity was inhibited, the number of surviving mycobacteria in infected cells significantly increased under multiple infection conditions. Significance: ns, not significant; ** P < 0.01; *** P <0.001.

[0055] Figure 3 The study showed that the proliferation of Mycobacterium tuberculosis was significantly reduced in HIF-2α-overexpressing macrophages. In this invention, an HIF-2α overexpression plasmid was constructed and transfected into macrophages. RT-qPCR confirmed a significant increase in HIF-2α mRNA levels. Mycobacterium tuberculosis was infected with HIF-2α-overexpressing macrophages and control macrophages at an MOI of 1. The results showed that the proliferation of Mycobacterium tuberculosis was significantly reduced in HIF-2α-overexpressing cells. Significance: ns, not significant; *** P < 0.001. Detailed Implementation

[0056] The following examples and experimental cases further illustrate the present invention and should not be construed as limiting the invention. The examples do not include detailed descriptions of conventional methods, such as methods for constructing vectors and plasmids, methods for inserting genes encoding proteins into vectors and plasmids, or methods for introducing plasmids into host cells. Such methods are well known to those skilled in the art and have been described in numerous publications.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention, and the preferred embodiments and materials described herein are for illustrative purposes only.

[0058] Example 1: Significantly increased HIF-2α expression levels in tuberculosis-associated monocytes and macrophages.

[0059] This example illustrates the expression changes of HIF-2α, namely EPAS1 / Epas1, in monocytes of tuberculosis patients and macrophages infected with mycobacteria.

[0060] 1. RNA-seq data analysis to detect the expression level of EPAS1 in mononuclear cells of tuberculosis patients.

[0061] Peripheral blood samples were collected from patients with active tuberculosis (TB) and healthy controls. Peripheral blood mononuclear cells were isolated, and total RNA was extracted and analyzed by RNA-seq. Sequencing data underwent quality control, alignment, expression quantification, and standardization. The expression difference of EPAS1 mRNA in mononuclear cells from patients with active TB and healthy controls was compared. Results showed that compared with healthy controls, the expression level of EPAS1 mRNA in mononuclear cells from patients with active TB was significantly increased, suggesting that HIF-2α is induced in TB-associated mononuclear cells.

[0062] 2. RT-qPCR validation of EPAS1 expression levels in an independent clinical cohort

[0063] To further validate the RNA-seq data, peripheral blood samples were collected from patients with active tuberculosis and healthy controls in an independent clinical cohort. Monocytes were isolated and total RNA was extracted. After obtaining cDNA via reverse transcription, the expression level of EPAS1 was detected using real-time quantitative PCR, and normalized analysis was performed using an internal reference gene.

[0064] The results showed that in an independent clinical cohort, the expression level of EPAS1 in monocytes of patients with active tuberculosis was also significantly higher than that in healthy controls, further confirming that HIF-2α expression was elevated in monocytes of tuberculosis patients.

[0065] 3. Mycobacterial infection induces increased expression of Epas1 in macrophages.

[0066] An in vitro mycobacterial infection model was established using immortalized bone marrow-derived macrophages (iBMDM). iBMDM cells were divided into an uninfected control group and a tuberculosis-infected group. In the tuberculosis-infected group, iBMDM cells were infected with an MOI of 10, and cell samples were collected at 0, 24, and 48 hours post-infection. Total RNA was extracted from the cells using TRIzol reagent, and cDNA was synthesized via reverse transcription. The expression level of the Epas1 gene was further detected using real-time quantitative PCR, and normalized analysis was performed using an internal control gene.

[0067] The results are as follows Figure 1 As shown. RNA-seq data showed that, compared with healthy controls, the expression level of EPAS1 mRNA in monocytes of patients with active tuberculosis was significantly increased ( Figure 1A). In an independent clinical cohort, RT-qPCR results further confirmed that EPAS1 expression levels in monocytes of patients with active tuberculosis were significantly higher than those in healthy controls. Figure 1 B). In the in vitro infection model, after Mycobacterium tuberculosis infection of iBMDM cells, the expression level of Epas1 was significantly increased at both 24 and 48 hours post-infection. Figure 1 C). The above results indicate that HIF-2α is upregulated in tuberculosis-associated monocytes and mycobacterial-infected macrophages.

[0068] Example 2: Inhibition of HIF-2α significantly increased the proliferation of mycobacteria in macrophages.

[0069] This example illustrates how knocking down small interfering RNA or inhibiting HIF-2α with small molecule inhibitors reduces the ability of macrophages to control mycobacterial proliferation.

[0070] 1. Knockdown of Epas1 expression in iBMDM cells using small interfering RNA

[0071] iBMDM cells were seeded at an appropriate density in cell culture plates. After the cells adhered and were in a suitable growth state, they were transfected with small interfering RNA targeting Epas1 to reduce the expression level of HIF-2α. A negative control group transfected with small interfering RNA was set up as a control group.

[0072] After transfection and culturing for an appropriate time, cell samples were collected. One portion of the cells was used to extract total RNA, and the expression level of EPas1 mRNA was detected by real-time quantitative PCR; the other portion of the cells was used to extract total protein, and the expression level of HIF-2α protein was detected by Western blotting.

[0073] The results showed that, compared with the negative control group, the level of Epas1 mRNA in the Epas1 small interfering RNA treatment group was significantly reduced, and the level of HIF-2α protein was reduced synchronously, indicating that small interfering RNA can effectively knock down HIF-2α expression in iBMDM cells.

[0074] 2. Detection of mycobacterial proliferation in macrophages after Epas1 knockdown

[0075] After confirming the effectiveness of Epas1 knockdown, Epas1-knockdown iBMDM cells were infected with Mycobacterium tuberculosis. After infection, free extracellular bacteria were removed, and the cells were cultured further. 48 hours post-infection, cells were lysed to release intracellular bacteria. The bacterial suspension was serially diluted and inoculated onto 7H10 medium and cultured at 32°C. Colony counting was performed after colony formation.

[0076] The results showed that, compared with the negative control group, the bacterial load in iBMDM cells of the Epas1 knockdown group was significantly increased, indicating that reducing HIF-2α expression weakens the ability of macrophages to restrict mycobacterial replication.

[0077] 3. Treatment of iBMDM cells with HIF-2α small molecule inhibitors

[0078] To further verify the effect of HIF-2α from a pharmacological perspective, iBMDM cells were treated with a small molecule inhibitor of HIF-2α. Cells were seeded at an appropriate density in 24-well cell culture plates. After cell attachment, culture medium containing the HIF-2α small molecule inhibitor PT2385 was added for 12 hours. A DMSO control group was also included.

[0079] 4. Detection of mycobacterial proliferation after HIF-2α inhibition under different infection multiplicity conditions

[0080] To investigate the effect of HIF-2α on the anti-mycobacterial function of macrophages under different infection intensities, iBMDM cells in the inhibitor-treated group and the DMSO control group were infected with Mycobacterium tuberculosis at MOI=1 and MOI=5. Cells were lysed 48 hours post-infection to release intracellular bacteria. The bacterial suspension was serially diluted and inoculated onto 7H10 medium and cultured at 32°C. Colony counting was performed after colony formation.

[0081] 5. Data statistical analysis

[0082] Statistical analysis software was used to analyze the experimental data. The t-test was used for comparisons between two groups, and analysis of variance was used for comparisons among multiple groups. A p-value less than 0.05 was considered statistically significant.

[0083] The results are as follows Figure 2 As shown. After transfecting iBMDM cells with small interfering RNA targeting Epas1, the level of Epas1 mRNA decreased significantly ( Figure 2 A), Immunoblotting results showed a synchronous decrease in HIF-2α protein levels ( Figure 2 B). Based on this, the above cells were infected with Mycobacterium tuberculosis and the number of intracellular bacteria was detected. The results showed that, compared with the control group, the bacterial load in macrophages of the Epas1 knockdown group was significantly increased ( Figure 2 C). Further treatment of iBMDM cells with a HIF-2α small molecule inhibitor significantly increased the number of surviving mycobacteria in infected cells under multiple infection multiplicity conditions. Figure 2 The above results indicate that HIF-2α plays an important role in limiting the proliferation of mycobacteria in macrophages, and that inhibiting HIF-2α leads to increased intracellular bacterial proliferation.

[0084] Example 3: HIF-2α overexpression inhibits mycobacterial infection

[0085] 1. Overexpression of HIF-2α in the iBMDM macrophage cell line

[0086] Cells were seeded at an appropriate density into 12-well cell culture plates, with 8 × 10⁶ cells per well. 5 Cells were cultured in an incubator for approximately 12 hours until they were fully adhered and entered the logarithmic growth phase. The overexpression plasmid pcDNA3.1-HIF2α (HIF-2α nucleotide sequence shown in SEQ ID NO. 1) was transfected into the cells using JetPEI. A control group (Vector group) transfected with an empty vector, pcDNA3.1-Vector, was also included. After 12 hours, the culture medium was replaced with complete medium, and the cells were cultured again for 48 hours to ensure adequate protein expression.

[0087] 2. Detect the expression level of the HIF-2α gene after overexpression.

[0088] Total RNA was extracted from cells using TRIzol reagent, and cDNA was synthesized via reverse transcription. Real-time quantitative PCR (qPCR) was employed, with specific primers used to detect the expression level of the HIF-2α gene EpasI, and β-actin was used as an internal reference gene for standardization.

[0089] 3. Bacterial proliferation assessment

[0090] Infection was performed using Mycobacterium tuberculosis, with an infection dose of 1 MOI. The plasmid-transfected iBMDM cells were inoculated into 24-well plates and cultured at 32°C with 5% CO2 after infection. Forty-eight hours post-infection, cells were lysed to release intracellular bacteria. The bacterial suspension was serially diluted and inoculated onto 7H10 medium and cultured at 32°C. Colony counting was performed after colony formation.

[0091] 4. Data Analysis

[0092] Data analysis was performed using statistical software (such as GraphPad Prism) to compare the proliferation of Mycobacterium tuberculosis in HIF-2α-overexpressing cells and control cells. The significance of differences between groups was assessed using t-tests, with a p-value <0.05 considered statistically significant.

[0093] The results are as follows Figure 3 As shown in the figure. First, qPCR results showed that the expression level of the EpasI gene was significantly upregulated in cells transfected with pcDNA3.1-HIF-2α plasmid ( Figure 3A) indicates that HIF-2α was successfully overexpressed and retained transcriptional activity in iBMDM cells. Further infection of macrophages in the HIF-2α overexpression group and the empty vector control group with Mycobacterium tuberculosis at MOI=1 showed that, compared with the empty vector control group, the proliferation of Mycobacterium tuberculosis in the HIF-2α overexpression group was significantly reduced (A). Figure 3 B). This result indicates that upregulating HIF-2α expression in macrophages can enhance the inhibitory effect of host cells on Mycobacterium tuberculosis, thereby reducing the proliferation of mycobacteria within the cells.

[0094] The nucleotide sequence encoding the HIF-2α protein is shown in SEQ ID NO. 1:

[0095]

[0096] The amino acid sequence of HIF-2α protein is shown as SEQ ID NO.2:

[0097] MTADKEKKRSSSERRKEKSRDAARCRRSKETEVFYELAHELPLPHSVSSHLDKASIMRLAISFLRTHKLLSSVCSENESEAEADQQMDNLYLKALEGFIAVVTQDGDMIFLSENISKFMGLTQVELTGHSIFDFTHPCDHEEIRENLSLKNGSGFGKKSKDMSTERDFFMRMKCTVTNRGRTVNLKSATWKVLHCTGQVKVYNNCPPHNSLCGYKEPLLSCLIIMCEPIQHPSHMDIPLDSKTFLSRHSMDMKFTYCDDRITELIGYHPEELLGRSAYEFYHALDSENMTKSHQNLCTKGQVVSGQYRMLAKHGGYVWLETQGTVIYNPRNLQPQCIMCVNYVLSEIEKNDVVFSMDQTESLFKPHLMAMNSIFDSSGKGAVSEKSNFLFTKLKEEPEELAQLAPTPGDAIISLDFGNQNFEESSAYGKAILPPSQPWATELRSHSTQSEAGSLPAFTVPQAAAPGSTTPSATSSSSSCSTPNSPEDYYTSLDNDLKIEVIEKLFAMDTEAKDQCSTQTDFNELDLETLAPYIPMDGEDFQLSPICPEERLLAENPQSTPQHCFSAMTNIFQPLAPVAPHSPFLLDKFQQQLESKKTEPEHRPMSSIFFDAGSKASLPPCCGQASTPLSSMGGRSNTQWPPDPPLHFGPTKWAVGDQRTEFLGAAPLGPPVSPPHVSTFKTRSAKGFGARGPDVLSPAMVALSNKLKLKRQLEYEEQAFQDLSGGDPPGGSTSHLMWKRMKNLRGGSCPLMPDKPLSANVPNDKFTQNPMRGLGHPLRHLPLPQPPSAISPGENSKSRFPPQCYATQYQDYSLSSAHKVSGMASRLLGPSFESYLLPELTRYDCEVNVPVLGSSTLLQGGDLLRALDQAT

[0098] The undescribed parts of this invention are the same as or implemented using existing technology. The applicant declares that this invention is illustrated through the above specific embodiments, but the invention is not limited to the above detailed methods, i.e., it does not mean that the invention must rely on the above detailed methods to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the product of this invention, additions of auxiliary components, and selection of specific methods all fall within the protection and disclosure scope of this invention.

Claims

1. Application of HIF-2α promoter in the preparation of drugs against mycobacterial infections.

2. The application according to claim 1, characterized in that, The anti-mycobacterial infection drug is selected from those used to treat mycobacterial infectious diseases and / or infection-related diseases and / or symptoms.

3. The application according to claim 2, characterized in that, The mycobacteria are selected from one or more of the following group: Mycobacterium tuberculosis, Mycobacterium marineum, avian intracellular mycobacteria complex, Mycobacterium kansas, Mycobacterium simianum, Mycobacterium bufota, Mycobacterium Gordon, Mycobacterium terrestrialum, and Mycobacterium paraterrestrialum. The diseases or symptoms associated with mycobacterial infection are selected from one or more of the following groups: histopathological damage caused by mycobacterial infection, insufficient or excessive production of cytokines after infection, inflammatory damage to organs, and multiple organ failure.

4. The application according to claim 1, characterized in that, The HIF-2α promoter is selected from any one or more of the following: (1) exogenous HIF-2α protein or nucleic acid encoding it; (2) liposomes, nanomaterials, PEG-modified proteins, protein microspheres or recombinant expression vectors that encapsulate nucleic acid molecules encoding HIF-2α protein.

5. The application according to claim 4, Its features are, The HIF-2α protein is selected from any of the following: (a) A polypeptide having the amino acid sequence shown in SEQ ID NO.2; (b) A substance that is homologous or identical to the amino acid sequence shown in SEQ ID NO.2 and that promotes HIF-2α function; (c) A substance in which one or more amino acids of (a) or (b) have been substituted, deleted, or added, and which promotes the function of HIF-2α, or a protein or polypeptide derived from (a) or (b), The nucleic acid molecule encoding the HIF-2α protein is selected from any of the following: (i) A nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO.1; (ii) Molecules that hybridize with the nucleotide sequence defined in (i) under strict conditions; (iii) Nucleic acid molecules that are homologous to or have the same sequence as the nucleotide sequence shown in SEQ ID NO.1; (iv) A nucleic acid molecule consisting of one or more nucleotides substituted, deleted or added from the nucleotide sequence in (i), (ii) or (iii).

6. The application according to claim 4, characterized in that, The recombinant expression vector encoding the HIF-2α protein nucleic acid molecule includes an expression vector and a nucleic acid molecule of the HIF-2α encoding gene EPAS1 inserted into the expression vector. The expression vector includes viral vectors and non-viral vectors.

7. An antibacterial agent for mycobacterial infection, characterized in that, Its active component includes the HIF-2α promoter as described in any one of claims 1 to 6.

8. The antimycobacterial infection preparation according to claim 7, characterized in that, This anti-mycobacterial infection agent is used in combination with other mycobacterial treatment drugs.

9. The application of reagents for detecting HIF-2α expression levels in the preparation of mycobacterial detection kits or antimicrobial drug screening kits, characterized in that, The kit contains primers that are specific for detecting the EPAS1 gene, and the primer sequences are shown in SEQ ID NO. 3 and 4.

10. A mycobacterial detection kit or an antimicrobial screening kit, the kit containing primers with detection specificity for the EPAS1 gene, the primer sequences of which are shown in SEQ ID NO. 3 and 4.

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