Application of TRIM58 gene in regulating host anti-mycobacterium bovis infection

Abstract

This invention discloses the application of the TRIM58 gene in regulating host resistance to Mycobacterium bovis infection. A bovine macrophage cell line with TRIM58 gene knockout was constructed using CRISPR / Cas9 technology. Results showed that TRIM58 knockout significantly inhibited intracellular survival of Mycobacterium bovis and reduced infection-induced cell death; reintroduction or overexpression of TRIM58 restored or enhanced bacterial viability. Therefore, this invention is the first to demonstrate that TRIM58 is a negative regulator of host resistance to Mycobacterium bovis infection, and that inhibitors of the TRIM58 gene can be used to prepare drugs against Mycobacterium bovis infection or to treat bovine tuberculosis. This invention fills a research gap in the field of TRIM58 in bovine tuberculosis and provides an effective tool for screening and studying the mechanisms of action of anti-bovine tuberculosis drugs.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and genetic engineering, specifically relating to the role of the TRIM58 gene in regulating host resistance to Mycobacterium bovis ( ). Mycobacterium bovis The present invention relates to the application of TRIM58 gene knockout in infection and the use of TRIM58 gene inhibitors in the preparation of anti-bovine tuberculosis drugs. The present invention also relates to a TRIM58 gene knockout genetically engineered cell and its construction method. Background Technology

[0002] Tuberculosis is an important zoonotic disease caused by the Mycobacterium tuberculosis complex (Mycobacterium tuberculosis complex). Mycobacterium tuberculosis complex Infections caused by Mycobacterium tuberculosis (Mycobacterium tuberculosis) Mycobacterium tuberculosis , M. tb ) and its bovine variant Mycobacterium bovis ( Mycobacterium bovis , M. bovis Bovine tuberculosis is a major source of human tuberculosis infection. Official policies worldwide mandate a "quarantine-culling" strategy to control bovine tuberculosis, which imposes a heavy economic burden on governments and practitioners in developing countries. Simultaneously, the increasing rate of bacterial drug resistance necessitates more effective control measures. Therefore, there is an urgent need in this field to develop new and effective methods for controlling tuberculosis.

[0003] In recent years, host-directed therapy (HDT) has attracted widespread attention as an emerging treatment strategy. This strategy enhances the host's immune capacity by targeting key host factors, thereby eliminating pathogens, including drug-resistant bacteria. In-depth research into the molecular mechanisms of host-pathogen interactions and the identification of key factors involved in host immune defense can provide target molecules for the development of new vaccines and drugs, as well as for cultivating disease-resistant strains through gene editing technology.

[0004] The TRIM (Tripartite Motif) protein family is one of the largest families of RING-type E3 ubiquitin ligases, containing over 80 members in the human genome. Members of this family possess a characteristic three-domain structure at the N-terminus: a RING finger domain (responsible for catalyzing ubiquitination), one or two B-box domains (mediating protein-protein interactions), and a coiled-coil domain (mediating dimerization and oligomerization); their C-terminal variable domain is responsible for substrate recognition. TRIM proteins are widely involved in various physiological processes, including cell proliferation, apoptosis, autophagy, and innate immune responses. Studies have shown that TRIM proteins can influence Mycobacterium tuberculosis infection through multiple pathways, including regulating autophagy and inflammatory responses.

[0005] In studies on the association between the TRIM family and tuberculosis, several members have been reported to participate in the regulation of mycobacterial infection. For example, a gene expression profiling study systematically analyzed the transcriptional levels of 72 TRIM genes in tuberculosis patients, finding that 20 TRIM genes were significantly downexpressed in patients with active tuberculosis. Among them, the expression levels of TRIM4, TRIM16, TRIM27, TRIM32, TRIM35, TRIM46, TRIM47, TRIM65, and TRIM68 were closely related to infection time and initial bacterial load. This study suggests that abnormal expression of TRIM genes may be involved in the pathogenesis of tuberculosis, and genes such as TRIM27 may serve as potential biomarkers indicating tuberculosis status.

[0006] Of particular note are the significant advances in recent years regarding the role of TRIM27 in Mycobacterium tuberculosis infection. In 2024, Zhao et al. revealed that TRIM27 is a protective factor against Mycobacterium tuberculosis infection. This study found that TRIM27 enters the cell nucleus after Mycobacterium tuberculosis infection and functions as a transcriptional activator of the transcription factor TFEB: TRIM27 directly binds to the TFEB gene promoter and interacts with the TFEB transcription factor CREB1, enhancing CREB1's binding affinity to the TFEB promoter, thereby promoting CREB1's transcriptional activity towards TFEB, ultimately inducing the expression of autophagy-related genes and activation of autophagic flux to clear the pathogen. This study also found that the TRIM27 gene transcription level in the peripheral blood of tuberculosis patients was significantly lower than that in healthy controls, suggesting that defective TRIM27 expression may be a potential cause of tuberculosis development. Furthermore, TRIM14 has also been reported to play a role in Mycobacterium tuberculosis infection by negatively regulating the expression of type I interferon in macrophages through TBK1 regulation of STAT3 phosphorylation.

[0007] In summary, TRIM family proteins are important host factors regulating Mycobacterium tuberculosis infection and are closely related to the occurrence and development of tuberculosis. However, the role of another important member of the TRIM family—TRIM58—in bovine Mycobacterium infection and bovine tuberculosis has not been reported domestically or internationally. Whether TRIM58 participates in the host's anti-mycobacterial immune response, its molecular mechanism of action, and whether it can serve as a target for the development of anti-bovine tuberculosis drugs and disease-resistant breeding all require further investigation. This invention fills this technological gap by revealing for the first time the function of the TRIM58 gene in regulating the host's resistance to bovine Mycobacterium infection and providing its application in the prevention and treatment of bovine tuberculosis. Summary of the Invention

[0008] The purpose of this invention is to address the lack of existing technology regarding the application of the TRIM58 gene in combating bovine mycobacterium infection, and to provide the application of TRIM58 gene inhibitors in the preparation of drugs for combating bovine mycobacterium infection or treating bovine tuberculosis.

[0009] To achieve the above objectives, this invention utilizes CRISPR / Cas9 gene editing technology to construct a targeting vector for the TRIM58 gene in the bovine macrophage (BoMac) cell line. Following vector transfection and cell screening, a TRIM58 gene knockout cell line and a cell model resistant to Mycobacterium bovis infection were established. The results showed that the TRIM58 gene knockout cells exhibited significant resistance to Mycobacterium bovis infection, and the intracellular survival of Mycobacterium bovis in the knockout cells was significantly lower than that in the wild-type cells, indicating that at the cellular level, TRIM58 gene knockout effectively inhibits Mycobacterium bovis infection; conversely, reintroduction or overexpression of the TRIM58 gene weakened the cells' anti-infection effect. These results demonstrate that the TRIM58 gene plays an important role in regulating host resistance to Mycobacterium bovis infection, confirming that the TRIM58 gene can be used as a target for the preparation of drugs against Mycobacterium bovis infection or for the treatment of bovine tuberculosis. This strategy can be applied to the preparation of TRIM58 gene-modified cells and animals resistant to Mycobacterium bovis infection, as well as the development of novel drugs.

[0010] Based on this, in a first aspect, the present invention provides the use of an inhibitor of the TRIM58 gene in the preparation of a drug for treating bovine mycobacterium infection or bovine tuberculosis, wherein the protein sequence encoded by the TRIM58 gene is shown in SEQ ID NO.1.

[0011] Furthermore, the inhibitors of the TRIM58 gene include: (1) sgRNA, siRNA, shRNA or antisense oligonucleotides targeting the TRIM58 gene; (2) CRISPR / Cas9 gene editing system for knocking out or silencing the TRIM58 gene; (3) Antibodies or antigen-binding fragments that specifically inhibit the expression of TRIM58 protein; (4) Small molecule compounds or natural product inhibitors.

[0012] According to a specific embodiment of the present invention, the inhibitor of the TRIM58 gene is a CRISPR / Cas9 gene editing system for knocking out or silencing the TRIM58 gene, comprising a Cas9 gene editing protein or its expression vector, and an sgRNA or its expression vector that guides the Cas9 gene editing protein to specifically bind to the TRIM58 gene.

[0013] The nucleotide sequence of the sgRNA is shown in SEQ ID NO:2.

[0014] Secondly, this invention provides a genetically engineered cell resistant to Mycobacterium bovis infection, wherein the TRIM58 gene in the cell is knocked out or silenced, giving it the potential to inhibit Mycobacterium bovis infection. The genetically engineered cell constructed by this invention exhibits significant resistance to Mycobacterium bovis infection and can be applied to high-throughput drug screening.

[0015] Thirdly, the present invention provides a method for constructing the aforementioned genetically engineered cells, comprising the following steps: (1) Construct a cell line that stably expresses Cas9 protein; (2) Design sgRNA and its primers targeting the TRIM58 gene, and construct sgRNA expression vector; (3) Package the sgRNA expression vector into a lentivirus and infect the cell line described in step (1); (4) Screening monoclonal cell lines from polyclonal cell lines; The nucleotide sequence of the sgRNA is shown in SEQ ID NO:2.

[0016] According to a specific embodiment of the present invention, the cell is a mammalian cell, including bovine macrophages.

[0017] According to a specific embodiment of the present invention, the primer sequences are shown in SEQ ID NO:3 and SEQ ID NO:3.

[0018] Compared with the prior art, the present invention has the following beneficial effects: This invention reveals for the first time the negative regulatory function of the TRIM58 gene in host resistance to Mycobacterium bovis infection, filling a research gap in the field of TRIM58 in bovine tuberculosis. It demonstrates that knocking out the TRIM58 gene significantly inhibits the survival of Mycobacterium bovis in host cells and reduces bacterial infection-induced cell death, while not affecting normal cell proliferation, indicating that TRIM58 is an ideal target for anti-tuberculosis drugs and disease-resistant breeding. This invention constructs a TRIM58 gene knockout bovine macrophage model, providing an effective tool for screening and studying the mechanisms of anti-bovine tuberculosis drugs. Attached Figure Description

[0019] Figure 1 Western blot analysis of Cas9 protein expression in bovine lung macrophage monoclonal cell lines.

[0020] Figure 2 Results of T7E1 restriction enzyme digestion assay for screening Cas9 monoclonal cell lines with high cleavage activity.

[0021] Figure 3 Sanger sequencing results of the TRIM58 gene knockout cell line showed a one-base insertion in the sgRNA target region.

[0022] Figure 4 Results of RT-qPCR detection of TRIM58 mRNA expression deficiency in TRIM58 gene knockout cell lines.

[0023] Figure 5 Western blot analysis confirmed the successful construction of TRIM58 overexpression and replenishment cell lines.

[0024] Figure 6 EdU cell proliferation assay to detect the effects of TRIM58 gene knockout, complementation, and overexpression on the proliferation capacity of bovine macrophages. A: Fluorescence microscopy; B: Quantitative analysis of EDU proliferation rate.

[0025] Figure 7 Effects of TRIM58 gene knockout, complementation, and overexpression on intracellular survival of Mycobacterium bovis (colony count results). p <0.05;** p <0.01; ***, p <0.001.

[0026] Figure 8 Effect of TRIM58 gene knockout on Mycobacterium bovis infection-induced cell death (cell viability as determined by CCK-8 assay). *** p <0.001. Detailed Implementation

[0027] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and not for limiting the scope of protection of the present invention. Various modifications or equivalent substitutions made by those skilled in the art based on the following embodiments should also be considered to fall within the scope of protection of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions or reference books such as *Molecular Cloning: A Laboratory Manual* (New York: Cold Spring Harbor Laboratory, 2017), or according to the methods recommended in the manufacturer's operating manual. Materials in the embodiments that do not specify their source are all commonly used materials well known in the art, which can be constructed based on literature reports or obtained commercially.

[0028] Key material descriptions: Lenti-Cas9-Blast is a lentiviral vector commonly used for gene editing in mammalian cells. Based on the CRISPR / Cas9 system, it expresses a humanized codon-optimized Cas9 protein and carries a blastidin resistance gene as a selection marker. Purchased from InvivoGen, catalog number ant-bl-05.

[0029] pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP: This is a lentiviral gRNA expression vector for CRISPR gene editing systems, featuring an optimized gRNA scaffold structure and dual labeling with puromycin (puro) and blue fluorescent protein (BFP) for easy screening and tracking of transduced cells. Purchased from Addgene, catalog number 67991.

[0030] pLVX-EGFP-IRES-Neo: Overexpression vector. Purchased from Addgene, catalog number 128660.

[0031] pMD19-T: Cloning vector. Purchased from Takara, catalog number 3271.

[0032] HEK293T cells: Lentiviral packaging cells. Purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number GNHu44.

[0033] Bovine lung macrophages (BoMac): Host cells used in experiments. Preserved in this laboratory.

[0034] Example 1: Construction and Phenotypic Verification of TRIM58 Gene Knockout Bovine Lung Macrophages 1. Construct a bovine lung macrophage cell line (BoMac-Cas9) expressing Cas9 protein with high cleavage activity. To obtain a stable bovine lung macrophage cell line expressing Cas9 protein, HEK293T cells were first co-transfected with lentiviral packaging helper plasmids pMD2.G and psPAX2 and Lenti-Cas9-Blast plasmid at a mass ratio of 1:2:3 for lentiviral packaging. Viral supernatant was collected and used to infect bovine lung macrophage (BoMac) cells. Polyclonal cell lines expressing Cas9 protein were obtained through selection with plasticidin. Monoclonal cell lines were further obtained using limiting dilution. Cas9 protein expression was detected by Western blot, and the results are shown below. Figure 1 As shown, multiple monoclonal cell lines successfully expressed the Cas9 protein.

[0035] To screen Cas9 monoclonal cell lines with high endonuclease activity, a specific sgRNA (sequence: 5'-GTGCACCAAGCAAACCCCAG-3') targeting the bovine MSTN gene exon was designed, and the sgRNA expression vector pKLV2-U6gRNA5-MSTN-KO was constructed. The Cas9 monoclonal cell lines were infected using a lentiviral packaging method. After 7 days of Puromycin selection, genomic DNA was extracted from the cells, the target region was amplified by PCR, and T7E1 restriction enzyme digestion was performed. The results showed ( Figure 2 Clone#1C9 showed the highest cleavage efficiency, reaching 52.5%, so the cell line was named BoMac-Cas9 for subsequent TRIM58 gene knockout experiments.

[0036] 2. Constructing a recombinant plasmid targeting the TRIM58 gene with sgRNA sgRNAs were designed using the sgRNAcas9 software targeting the TRIM58 gene sequence. Primers were designed based on sgRNAs targeting gene exons, and the designed primers were sent to a company for synthesis. The sequences are as follows: TRIM58-sgRNA: 5'-GGAACCCGAGTCCATCGTTA-3'.

[0037] TRIM58-sgR-F: 5'-CACCGGGAACCCGAGTCCATCGTTA-3', TRIM58-sgR-R: 5'-AAACTAACGATGGACTCGGGTTCCC-3'.

[0038] After annealing the primers, they were ligated into the BsmBI-linearized pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP vector, transformed into E. coli, and positive clones were selected for Sanger sequencing verification. Colonies of positive clones were selected for expansion culture, and plasmids were extracted using an endotoxin-free kit. The constructed recombinant plasmid was named pKLV2-U6gRNA5-TRIM58-KO.

[0039] 3. Preparation of TRIM58 gene knockout cell lines HEK293T cells were co-transfected with pMD2.G, psPAX2, and pKLV2-U6gRNA5-TRIM58-KO plasmid at a mass ratio of 1:2:3 to package lentivirus. Viral supernatant was collected and used to infect BoMac-Cas9 monoclonal cell lines, followed by 7 days of Puromycin selection. Genomic DNA was then extracted from the cells, and amplification primers (TRIM58-PCR-F: 5'-AAGAGATGTTCCTGGTGTGTCC-3', TRIM58-PCR-R: 5'-CTGCCAGATAACGAAAGAATCC-3') were designed for PCR amplification. The product was cloned into the pMD19-T vector, and single clones were selected for Sanger sequencing. Furthermore, total RNA was extracted from cells using the Trizol method, and the transcriptional expression level of TRIM58 in monoclonal cells was detected using RT-qPCR. The primer sequences for relative quantitative qPCR were TRIM58-QF: 5'-GAGCCTTGAGCCGAAGTAAGA-3' and TRIM58-QR: 5'-GGGACATCCCTCCACAGTTC-3'.

[0040] Sequencing results showed ( Figure 3 Compared to wild-type cells, TRIM58-KO#152 monoclonal cells had a one-base insertion in the sgRNA targeting region, resulting in a frameshift mutation in the coding sequence. RT-qPCR results showed that... Figure 4 The TRIM58 mRNA expression was absent in this cell line. Thus, a TRIM58 gene knockout cell line was successfully constructed and named TRIM58-KO.

[0041] Example 2: TRIM58 gene regulates host resistance M. bovis Functional evaluation To evaluate the role of the TRIM58 gene in host resistance to Mycobacterium bovis ( M. bovis To assess the function of TRIM58 gene knockout, complementation, and overexpression cell lines during infection, this embodiment first constructed TRIM58 gene overexpression and complementation cell lines, and then evaluated the function of TRIM58 gene knockout, complementation, and overexpression cell lines from three aspects: cell proliferation, bacterial intracellular survival, and cell death.

[0042] 1. Construction of TRIM58 gene overexpression and complementation cell lines 1.1 Construction of overexpression and complementation plasmids The target fragment was synthesized by Beijing Qingke Biotechnology Co., Ltd. based on the coding sequence (CDS) of the TRIM58 gene. A flag tag was added before the stop codon at the end of the target sequence, and the fragment was cloned into the pLVX-EGFP-IRES-Neo vector to construct an overexpression plasmid named pLVX-TRIM58-flag-OE.

[0043] To construct the complement plasmid, during the synthesis of the CDS region of the TRIM58 gene, the amino acid near the NGG position of the sgRNA was mutated to a synonymous amino acid (i.e., the nucleotide sequence was changed without altering the amino acid sequence, so that it could no longer be recognized by the sgRNA), thus obtaining the TRIM58 gene complement sequence. This was then cloned into the pLVX-EGFP-IRES-Neo vector to construct the complement plasmid, named pLVX-TRIM58-flag-CO. The endotoxin-free plasmid was extracted using a plasmid miniprep kit (Omega Bio-Tek; catalog number: D6950-02) and stored at -80℃ for later use.

[0044] 1.2 Lentiviral Packaging and Cell Infection Referring to the lentivirus packaging method in Example 1, the packaging helper plasmids pMD2.G and psPAX2 were co-transfected into HEK293T cells with the above-mentioned pLVX-TRIM58-flag-OE overexpression plasmid or pLVX-TRIM58-flag-CO complement plasmid at a mass ratio of 1:2:3 to perform lentivirus packaging. The viral supernatant was collected for later use.

[0045] Wild-type BoMac cells and TRIM58 knockout cells (KO TRIM58) were revived in six-well plates. When the cell density reached approximately 60%, the old culture medium was discarded, and 1 mL of cell maintenance medium (DMEM containing 2% FBS and 1% penicillin antibody) was added to each well. Then, 1 μL of polybrene and 40 μL of the corresponding lentivirus were added (overexpressing lentivirus for wild-type BoMac cells and replacement lentivirus for KO TRIM58 cells). After mixing, the cells were incubated for 24 hours. The old culture medium was then discarded, and 2 mL of DMEM containing 5% FBS and 1% penicillin antibody was added. Incubation was continued for another 24 hours. Subsequently, the medium was replaced with DMEM selection medium containing 400 μg / mL neomycin (containing 5% FBS and 1% penicillin antibody) for drug screening. The selection medium was changed every 2-3 days. Drug screening continued for 7 days. After all negative control cells died, the TRIM58 overexpressing cell line (OE TRIM58) and the TRIM58 replacement cell line (CO TRIM58) were obtained.

[0046] 1.3 Validation of overexpression and replacement cell lines Total protein was extracted from both overexpressing and replacement cells, and Western blot was used to verify the success of the overexpression and replacement experiments. Results are as follows: Figure 5 As shown: no TRIM58-Flag fusion protein expression was detected in wild-type BoMac cells expressing the empty vector; a clear Flag tag signal was detected in the overexpression cell line (OE TRIM58), indicating successful overexpression of the TRIM58 protein; a Flag tag signal was also detected in the complement cell line (CO TRIM58), indicating successful complement expression of the exogenous TRIM58 gene in the TRIM58 gene knockout cells. These results indicate that the TRIM58 overexpression and complement cell lines were successfully constructed and can be used for subsequent functional evaluation experiments.

[0047] 2. Effects of TRIM58 gene knockout on cell proliferation The effect of TRIM58 gene knockout on the normal proliferation capacity of BoMac cells was evaluated using an EdU cell proliferation assay. An equal volume of 2× EdU working solution (20 μM) preheated to 37°C was added to a cell culture plate and mixed with the existing culture medium to achieve a final EdU concentration of 1×. The plate was incubated for 2 hours. Subsequently, the cells were fixed with pre-cooled 4% paraformaldehyde at room temperature for 15 minutes, washed with PBS, permeabilized with 0.3% Triton X-100 at room temperature for 10 minutes, washed again, and then incubated with Click reaction solution at room temperature in the dark for 30 minutes. After washing three times with PBS, the cells were incubated with DAPI staining solution in the dark for 10 minutes, and observed and photographed under a fluorescence microscope.

[0048] The results are as follows Figure 6 As shown, compared with wild-type (WT) cells, there was no significant difference in the proportion of EdU-positive cells among TRIM58 gene knockout cells (KO TRIM58), complement cells (CO TRIM58), and overexpression cells (OE TRIM58) (p>0.05), indicating that knockout, complementation, or overexpression of the TRIM58 gene does not affect the normal proliferative capacity of bovine macrophages.

[0049] 3. Knocking out the TRIM58 gene M. bovis Effects on intracellular survival With 1×10 per hole 5 Wild-type BoMac cells, TRIM58 knockout cells (KOTRIM58), complement cells (CO TRIM58), and overexpressing cells (OE TRIM58) were seeded in 12-well plates at specific ratios and cultured at 37°C and 5% CO2 until adherent. Cells were washed three times with 1640 incomplete culture medium before infection. M. bovisAfter washing and dispersing with HBSS, cells were inoculated into 12-well plates at a multiplicity of infection (MOI) of 10:1 and incubated at 37°C for 2 hours. After infection, the supernatant was discarded, and the cells were washed three times with 1640 incomplete medium. 1 mL of 1640 incomplete medium containing 50 μg / mL gentamicin (containing 5% FBS) was added to each well, and the cells were treated for 2 hours to kill extracellular bacteria. The cells were then washed three times with 1640 incomplete medium, and 1 mL of 1640 medium containing 5% FBS was added for further incubation, recorded as 0 hours post-infection. Cells were lysed at 0 h, 24 h, 48 h, and 72 h post-infection, and the lysate was plated on 7H11 plates at appropriate dilutions. After incubation at 37°C for approximately 3 weeks, colony counts (CFU) were performed.

[0050] The results are as follows Figure 7 As shown: Compared with wild-type cells, TRIM58 gene knockout cells M. bovis The number of intracellular surviving bacteria was significantly reduced; however, after TRIM58 gene reintroduction, bacterial intracellular survival was restored, and overexpression of the TRIM58 gene further increased bacterial intracellular survival. These results indicate that TRIM58 gene knockout can significantly inhibit... M. bovis Survival within the host cell is crucial, while expression of the TRIM58 gene promotes intracellular bacterial survival.

[0051] 4. Knocking out the TRIM58 gene M. bovis Effects of induced cell death To evaluate the effects of TRIM58 gene knockout on M. bovis The effect of infection-induced cell death, in wild-type BoMac cells and TRIM58 knockout cells (KO TRIM58) infected with a lethal dose of MOI=20, in M. bovis After 24h, 48h, 72h, and 96h of infection, 10 μL of CCK-8 solution was added to each well, and the cells were incubated at 37℃ for 2 hours. The absorbance at 450 nm was measured using a microplate reader, and the cell viability was calculated.

[0052] The results are as follows Figure 8 As shown, in M. bovis Following infection, the survival rate of wild-type cells was significantly reduced, while the survival rate of TRIM58 gene knockout cells was significantly higher than that of wild-type cells. This indicates that TRIM58 gene knockout can effectively alleviate the risk of infection. M. bovis Infection-induced host cell death enhances the host cell's tolerance to bacterial infection.

[0053] In conclusion, TRIM58 gene knockout does not affect the normal proliferative capacity of bovine macrophages, but it can significantly inhibit [the proliferation of macrophages]. M. bovisThe TRIM58 gene helps bacteria survive within host cells and mitigates infection-induced cell death; conversely, recombinant or overexpression of the TRIM58 gene restores or enhances intracellular survival. These results strongly suggest that the TRIM58 gene plays a crucial role in regulating host resistance. M. bovis Key negative regulators of infection can serve as important targets for the development of anti-bovine tuberculosis drugs and disease-resistant breeding.

Claims

1. An inhibitor of the TRIM58 gene in the preparation of a drug for treating Mycobacterium bovis ( ). Mycobacterium bovis The TRIM58 gene is used in drugs for the infection or treatment of bovine tuberculosis, and the protein sequence encoded by the TRIM58 gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The inhibitors of the TRIM58 gene include: (1) sgRNA, siRNA, shRNA or antisense oligonucleotides targeting the TRIM58 gene; (2) CRISPR / Cas9 gene editing system for knocking out or silencing the TRIM58 gene; (3) Antibodies or antigen-binding fragments that specifically inhibit the expression of TRIM58 protein; (4) Small molecule compounds or natural product inhibitors.

3. The application according to claim 2, characterized in that: The CRISPR / Cas9 gene editing system comprises the Cas9 gene editing protein or its expression vector, and the sgRNA or its expression vector that guides the Cas9 gene editing protein to specifically bind to the TRIM58 gene.

4. The application according to claim 3, characterized in that: The nucleotide sequence of the sgRNA is shown in SEQ ID NO:

2.

5. A genetically engineered cell resistant to Mycobacterium bovis infection, characterized in that, The TRIM58 gene in the cells was knocked out or silenced, which inhibited the survival of Mycobacterium bovis in the cells and reduced cell death. The protein sequence encoded by the TRIM58 gene is shown in SEQ ID NO.

1.

6. A method for constructing the genetically engineered cell of claim 5, characterized in that, Includes the following steps: (1) Construct a cell line that stably expresses Cas9 protein; (2) Design sgRNA and its primers targeting the TRIM58 gene, and construct sgRNA expression vector; (3) Package the sgRNA expression vector into a lentivirus and infect the cell line described in step (1); (4) Screening monoclonal cell lines from polyclonal cell lines; The nucleotide sequence of the sgRNA is shown in SEQ ID NO:

2.

7. The method according to claim 6, characterized in that, The cells are mammalian cells, including bovine macrophages.

8. The method according to claim 6, characterized in that, The primer sequences are shown in SEQ ID NO:3 and SEQ ID NO:3.

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