Application of NmrAL2_Bovine gene as a target in the prevention and treatment of bovine tuberculosis

Abstract

This invention discloses NmrAL2_Bovine The application of genes as targets in the prevention and control of bovine tuberculosis, including knockout or knockdown. NmrAL2_ Bovine The gene can significantly inhibit the survival of Mycobacterium bovis, Mycobacterium tuberculosis, Mycobacterium bovis BCG, and Mycobacterium smegmae in host cells without affecting the normal proliferation of host cells. Therefore, NmrAL2_Bovine Gene inhibitors can be used to prepare drugs for the prevention and treatment of bovine tuberculosis. NmrAL2_Bovine Genes can also be used as targets in the design of transgenic cattle for breeding against bovine tuberculosis or for genetic therapy research.

Description

Technical Field

[0001] This invention belongs to the field of animal disease resistance technology, specifically involving the application of the NmrAL2_Bovine gene as a target in the prevention and treatment of bovine tuberculosis. Background Technology

[0002] Tuberculosis is an important zoonotic disease caused by infection with the Mycobacterium tuberculosis complex, primarily including *Mycobacterium tuberculosis* (M.tb) and its bovine variant, *Mycobacterium bovis* (M.bovis). The World Organisation for Animal Health (WOAH) lists animal tuberculosis as a reportable disease, and my country classifies bovine tuberculosis as a Class II animal disease and one of the eight key zoonotic diseases requiring prevention and control. Because bovine tuberculosis is a significant source of infection for human tuberculosis, it is crucial for food safety and public health, and countries worldwide officially mandate a "quarantine-culling" strategy to control it. Simultaneously, the enormous burden of human tuberculosis and the increasing rate of bacterial resistance urgently require more effective control measures. Therefore, there is an urgent need in this field to develop a new and effective method for controlling zoonotic tuberculosis.

[0003] Nitric oxide (NO), as an important molecule, plays a crucial role in the regulation of tuberculosis. Moderate NO production can kill pathogenic microorganisms, while excessive NO production leads to an overactive inflammatory response, causing cell damage and ultimately cell death. Although Nitrogen metabolite repressor (NmrA) and most NmrA-like proteins have been identified in bacteria or fungi, there is also a widely distributed NmrA-like protein in higher animals, namely NMRAL1, also known as HSCRG. In the cytoplasm, NMRAL1 negatively regulates innate immunity by inhibiting the activity of the NF-κB and RLR pathways. NMRAL1 acts as a link between cellular redox state and other signaling pathways, inhibiting NO production by binding to Argininosuccinate synthetase (AS). ENSBTAG00000011305 is a bovine gene with unknown function. Gene family analysis revealed that this gene contains an NmrA domain, belonging to the NmrA family of proteins. Therefore, we named this gene NmrAL2_Bovine. Overall, the functions of NmrA family proteins in infectious diseases and immune regulation make it an important area for future research. However, to date, the role of NmrAL2_Bovine in the pathogenesis of bovine tuberculosis has not been reported, and its mechanism of regulating mycobacterial infection requires further investigation. Summary of the Invention

[0004] This invention protects, in one aspect, the use of an NmrAL2_Bovine gene inhibitor—for the preparation of a drug to prevent or treat bovine tuberculosis. Further, the NmrAL2_Bovine gene inhibitor comprises siRNA that targets and knocks down the expression of the NmrAL2_Bovine gene, or a nucleic acid molecule or expression vector thereof that targets and knocks out the NmrAL2_Bovine gene. Preferably, the targeting sequence of the siRNA is 5'-UGGCUUCUGCUAAGCUACCUCUGAG-3'. Preferably, the sequence of the nucleic acid molecule that targets and knocks out the NmrAL2_Bovine gene is 5'-GGTATCTCGGTTGCTGATAT-3'.

[0005] This invention protects, in one aspect, drugs for the preparation of prevention or treatment of bovine tuberculosis, including nucleic acid molecules or expression vectors that target and knock out the NmrAL2_Bovine gene, preferably with the sequence 5'-GGTATCTCGGTTGCTGATAT-3'. It also includes nucleic acid molecules that target and knock down the NmrAL2_Bovine gene, namely, double-stranded RNA hybridized to the NmrAL2_Bovine gene, wherein the sequence of the first strand of the double-stranded RNA is identical to the target sequence in the NmrAL2_Bovine gene, and the target sequence is 5'-UGGCUUCUGCUAAGCUACCUCUGAG-3'.

[0006] In another aspect, this invention protects a cell model resistant to bovine tuberculosis, wherein the endogenous NmrAL2_Bovine gene in the cell model is knocked out or knocked down, thereby resulting in the absence or reduction of expression of the endogenous NmrAL2_Bovine gene. The successfully constructed NmrAL2_Bovine gene knockout cell line is named fetal bovine lung cell EBL / NmrA-KO#9 (abbreviated as NmrAL2-KO), and this cell line was deposited on November 14, 2023, at the China Center for Type Culture Collection (CTCC), Wuhan University, Wuhan, China, accession number C2023350.

[0007] Another aspect of this invention protects a method for preparing gene-edited cattle resistant to bovine tuberculosis, which involves knocking out or knocking down the NmrAL2_Bovine gene in bovine cells, thereby causing the expression of the endogenous NmrAL2_Bovine gene to be absent or reduced, and further preparing gene-edited cattle with NmrAL2_Bovine gene knockout or knockdown.

[0008] The present invention also protects a method for screening candidate compounds for the prevention and / or treatment of bovine tuberculosis, comprising setting up a test group and a control group, wherein the test group is in which the test compound is added to cells, and the control group is in which the test compound is not added to cells, comparing the expression level of the NmrAL2_Bovine gene in the cells of the test group and the control group, and screening for test compounds that significantly reduce the expression level of the NmrAL2_Bovine gene.

[0009] The technical solution of the present invention has the following main beneficial effects:

[0010] 1. This invention provides a novel application of the NmrAL2_Bovine inhibitor for inhibiting the survival of Mycobacterium tuberculosis complex in host cells, thereby resisting bovine tuberculosis caused by Mycobacterium tuberculosis complex infection.

[0011] 2. This invention provides a genetically engineered cell line resistant to Mycobacterium tuberculosis complex infection and its preparation method. The cell line can resist Mycobacterium tuberculosis complex infection without affecting normal cell function, and can be used as an in vitro experimental model, reducing the impact of chemical additives on cell experiments.

[0012] 3. This invention provides a method for preparing gene-edited cattle resistant to bovine tuberculosis, and uses this method to prepare a bovine model resistant to bovine tuberculosis for research on anti-tuberculosis drugs or preparations.

[0013] 4. This invention provides a method for screening candidate compounds for the prevention and / or treatment of tuberculosis, using the NmrAL2_Bovine gene as a target to screen test compounds that significantly reduce the expression level of the NmrAL2_Bovine gene. Attached Figure Description

[0014] Figure 1 Identification of NmrAL2_Bovine gene knockout cell lines. Figure 1 In Example 1, A represents the genotype of the NmrAL2_Bovine knockout cell line detected by Sanger sequencing. WT represents wild type. NmrAL2-KO represents NmrAL2_Bovine gene knockout cells constructed using the CRISPR / Cas9 lentivirus strategy. Del 12 represents the deletion of 12 bases. PAM is an abbreviation for protpspacer adjacent motif. sgRNA is an abbreviation for small guide RNA. Figure 1 In Figure B, the transcription level of the NmrAL2_Bovine gene in the NmrAL2-KO cell line in Example 1 was detected by RT-qPCR. Figure 1In Figure C, the transcriptional level of the NmrAL2_Bovine gene after siRNA treatment was detected by RT-qPCR in Examples 3 and 4. si-NmrAL2 represents the interfering RNA group, and NC siRNA represents the negative control group. T-tests were used: ns, p>0.05**, p<0.01; ***, p<0.001.

[0015] Figure 2 In Example 2, the effect of NmrAL2_Bovine gene knockout on EBL cell proliferation was detected using CCK8 assay. WT-EBL represents wild-type EBL cells, and NmrAL2-KO represents NmrAL2_Bovine gene knockout EBL cells. A t-test was used, and ns, p>0.05 were found.

[0016] Figure 3 In Example 3, CFU counting was used to detect the intracellular viability of *M. bovis*, *M. tb*, *M. bovis BCG*, and *M. smegmatis* after infection in NmrAL2_Bovine knockout (AD) and knockdown (EH) cells. WT-EBL represents wild-type EBL cells, NmrAL2-KO represents NmrAL2_Bovine gene knockout cells constructed using a CRISPR / Cas9 lentiviral strategy, si-NmrAL2 represents NmrAL2_Bovine gene knockdown expression cells constructed using RNAi technology, and NC siRNA served as the negative control group. Analysis of variance was used; ns, p>0.05; *, p<0.05; **, p<0.01; ***, p<0.001.

[0017] Figure 4 In Example 4, the cell viability of NmrAL2_Bovine gene knockout (A, B) and knockdown (C, D) cells after infection with M. bovis and M. tb was detected using CCK8 assay. Analysis of variance was used, ns, p>0.05; *, p<0.05; **, p<0.01; ***, p<0.001.

[0018] Figure 5 The values ​​represent the intracellular NO concentration levels of wild-type cell lines EBL and NmrAL2_Bovine gene knockout cell line NmrAL2-KO after infection with Mycobacterium bovis in Example 5. ** indicates p<0.01. Detailed Implementation

[0019] Through extensive and in-depth research, the inventors have discovered for the first time that editing the NmrAL2_Bovine gene inhibits the intracellular survival of *M. bovis* in host cells and reduces infection-induced host cell death. Using CRISPR / Cas9 technology, an NmrAL2_Bovine gene knockout cell line was constructed. Infecting these cells with *M. bovis* and *M. tb* strains revealed that knocking out the NmrAL2_Bovine gene significantly reduced the survival of *M. bovis* and *M. tb* in host cells and infection-induced host cell death, thus providing a novel molecular target for inhibiting the intracellular survival of the *Mycobacterium tuberculosis* complex.

[0020] 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 only used to explain the present invention and not to limit 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 implemented under conventional conditions or according to the methods recommended in the manufacturer's operation manual. Materials whose sources are not specified in the embodiments, such as EBL cells, Cas9 protein, HEK293FT cells, lentiviral packaging plasmids, helper plasmids, T vectors, etc., are all commonly used materials well known in the art and can be constructed by oneself according to literature reports or obtained through commercial means. Specific information on plasmids used in this experiment can be found at http: / / www.addgene.org / , including Cas9 plasmid (#52962), enzyme digestion plasmid pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP (#67991), packaging helper plasmids pMD2.G (#12259), psPAX2 (#12260), etc.

[0021] Example 1. Construction of bovine NmrAL2_Bovine gene knockout cell line using CRSPR / Cas9 lentivirus strategy

[0022] 1.11 Design and Construction of Specific sgRNA Targeting the NmrAL2_Bovine Gene

[0023] sgRNAs targeting the exon sequence of the NmrAL2_Bovine gene (Ensembl: ENSBTAG00000011305) were designed using sgRNAcas9 software (https: / / sourceforge.net / projects / sgrnacas9 / ). Based on specificity evaluation results, sgRNAs targeting the bovine NmrAL2_Bovine gene exons were selected. The selected sgRNA sequence is: NmrAL2_Bovine-sgRNA: 5'-GGTATCTCGGTTGCTGATAT-3', where the PAM sequence recognizing the target is "TGG". The primer pairs for synthesizing the sgRNA were: NmrAL2_Bovine-sgR-F: 5'-CACCGGTATCTCGGTTGCTGATAT-3', and NmrAL2_Bovine-sgR-R: 5'-AAACATATCAGCAACCGAGATACC-3'. Subsequently, 5 μL each of the synthesized sgRNA primers F and R (10 pmol) were annealed in a PCR instrument: 95℃ for 10 min; 65℃ for 60 min. The annealed product was then ligated into the BsmBI (NEB) linearized pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP vector (Addgen#67991). Further transformation, plating, and overnight culture were performed. Single colonies were picked the next day for Sanger sequencing identification. Positive clones were selected for expansion culture, and plasmids were extracted using an endotoxin removal kit. The vector with correct sgRNA expression was named pKLV2-U6gRNA5-NmrAL2_Bovine-KO.

[0024] 1.2 Preparation of NmrAL2_Bovine gene knockout cell lines

[0025] HEK293FT cells were transfected with lentivirus at a mass ratio of pMD2.G:psPAX2:pKLV2-U6gRNA5-NmrAL2_Bovine-KO of 1:2:3 for lentivirus packaging. Subsequently, the cells were infected with the lentivirus to infect the bovine lung epithelial cell line EBL-Cas9, which expresses highly cleavable Cas9 protein (method described in CN118340888A). After 7 days of screening with Puromycin, genomic DNA was extracted from the cells using the DNA extraction kit (KG203) from Tiangen Biotech (Beijing) Co., Ltd. Targeting the NmrAL2_Bovine sgRNA target region in the genome, amplification primer pairs were designed using NCBI-BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi): NmrAL2_Bovine-PCR-F: 5'-AGCGACTCAGTGTTACATGGTG-3', NmrAL2_Bovine-PCR-R: 5'-TTCACCCCAACTCTGCTGTG-3'. Then, PCR was performed using genomic DNA as a template. Further, the purified PCR product was cloned into the pMD19-T vector, transformed, plated, and cultured overnight. Single colonies were picked for Sanger sequencing to identify the genotype of the monoclonal cells. The results showed that, compared with wild-type cells, the NmrAL2-KO monoclonal cells had a 12-base deletion in the sgRNA target region of the genome. Figure 1 (A) indicates a frameshift mutation in the coding sequence of the NmrAL2_Bovine gene. Further, mRNA was extracted, and the transcriptional expression of NmrAL2_Bovine in NmrAL2-KO cells was detected using RT-qPCR. Figure 1 As shown in Figure B, compared with wild-type cells, the NmrAL2_Bovine transcription level was significantly reduced in NmrAL2-KO knockout cells, indicating that the NmrAL2_Bovine knockout cell line was successfully constructed and named fetal bovine lung cell E BL / NmrA-KO#9 (abbreviated as NmrAL2-KO). This cell line was deposited at the China Center for Type Culture Collection on November 14, 2023, at Wuhan University, Wuhan, China, with accession number CCTCC NO: C2023350.

[0026] Example 2. Knocking out the NmrAL2_Bovine gene does not affect normal cell proliferation.

[0027] The effect of NmrAL2_Bovine gene knockout on EBL cell proliferation was evaluated using the CCK-8 assay. The experimental procedure was as follows: NmrAL2-KO cells were seeded into 96-well culture plates, and cell proliferation was detected using CCK-8 at 6, 12, 24 and 48 h: (1) Discard the old culture medium in the cell wells and replace it with fresh culture medium; (2) Add 1 / 10 volume of CCK-8 solution to the cell wells; (3) Incubate the culture plate in an incubator for 1 h; (4) Carefully aspirate the supernatant into the microplate, avoiding the generation of air bubbles; (5) Measure the absorbance at 450 nm using a microplate reader and calculate cell proliferation.

[0028] like Figure 2 As shown, CCK8 assay revealed no significant difference in growth rate between wild-type EBL cells and NmrAL2-KO cells at 6, 12, 18, and 24 hours, indicating that knockout of the NmrAL2 gene does not affect cell proliferation rate.

[0029] Example 3. Knockout and knockdown of the NmrAL2_Bovine gene significantly inhibited the survival of M. bovis, M. tb, M. bovis BCG, and Mycobacterium smegmatis in bovine lung epithelial cells.

[0030] With 3×10 per hole 5 Wild-type cell line EBL and knockout cell line NmrAL2-KO were seeded in 12-well plates at a ratio of 1:1 and cultured at 37°C and 5% CO2 until adherence. Before infection, the cells were washed three times with DMEM incomplete medium. The mycobacteria used for infection (M. bovis, M. tb, M. bovis BCG, and Mycobacterium smegmatis) were washed with HBSS and dispersed, then seeded into 12-well plates at a multiplicity of infection (MOI) of 10:1 to ensure uniform bacterial contact with the cells. The 12-well plates were incubated at 37°C for 6 hours in a 5% CO2 incubator. During this period, the initial bacterial culture was serially diluted 10-fold, and colonies were counted at appropriate dilutions. After infection, the supernatant was discarded, and the cells were washed three times with DMEM incomplete medium. 2 mL of DMEM incomplete medium (containing 2% FBS and 50 μg / mL gentamicin) was added to each well, and the plates were incubated for 1 hour to kill any remaining extracellular bacteria. After antibiotic treatment, cells were washed three times with DMEM incomplete medium, and then 1 mL of DMEM medium (containing 2% FBS) was added to each well for further incubation. This was recorded as 0 h post-infection. Subsequently, cells were lysed at different time points according to experimental requirements, and colony counts were performed using appropriately diluted lysates. The lysed cell solution was repeatedly pipetted to ensure homogeneity, and then 100 μL was continuously diluted 10-fold. Appropriately diluted lysates were then evenly spread onto 7H11 plates and incubated at 37°C for approximately 3 weeks before colony counting. Figure 3As shown in Figures A, B, C, and D, compared with the wild-type control group, the intracellular survival of M. bovis, M. tb, M. bovis BCG, and Mycobacterium smegmatis was significantly reduced in NmrAL2-KO.

[0031] Use EBL cells passaged to passages 3-10 for experiments. Transfection is performed when cells reach 70%-80% confluency. Prepare 1.5 mL RNase-free EP tubes, add 150 μL of opti-MEM medium to each tube, add Lipofectamine 2000, and time for 5 minutes. Label this tube A. Add the siRNA to be transfected to another tube, label this tube B. After 5 minutes, gently add the liquid from tube B dropwise to tube A, gently tapping the bottom to mix. Incubate at room temperature for 20 minutes to form the RNA-transfection reagent mixture (turbidity is normal and will not affect transfection). Remove the cell culture plate, wash the cells to prevent serum in the DMEM complete medium from affecting transfection; wash once with serum-free DMEM medium, then add opti-MEM medium. After 20 minutes, discard three wells and add three more wells, completing the process sequentially. Slowly add the transfection mixture dropwise to maximize transfection efficiency. After transfection, the cell culture plates were placed in a cell culture incubator at 37°C and 5% CO2. After 4-6 hours, the cells were washed once with serum-free DMEM medium, then serum-free DMEM medium was added again, and the cells were cultured for another 18-24 hours before infection. Samples were taken at different time points after infection. In the experiment, the transfection concentration of siRNA in both the negative control (NC siRNA) and the interfering RNA group was 100 nM. The sequence of the interfering siRNA targeting NmrAL2_Bovine was 5'-UGGCUUCUGCUAAGCUACCUCUGAG-3', the synthesized sense strand was 5'-UGGCUUCUGCUAAGCUACCUCUGAG-3', and the antisense strand was 5'-CUCAGAGGUAGCUUAGCAGAAGCCA-3'. Figure 1 As shown in Figure C, the expression of the NmrAL2_Bovine gene was successfully knocked down, and the constructed NmrAL2_Bovine gene knockdown expression cell was named si-NmrAL2. Further infection experiments demonstrated that knocking down NmrAL2_Bovine gene expression significantly reduced the intracellular survival of *M. bovis*, *M. tb*, *M. bovis BCG*, and *Mycobacterium smegma* in EBL cells. Figure 3 (E, F, G, H).

[0032] Example 4. Cells with knockout and knockdown of the NmrAL2_Bovine gene grew normally but were resistant to M. bovis and M. tb-induced cell death.

[0033] Infection of EBL cells with certain doses of *M. bovis* and *M. tb* can lead to cytopathic effects and even induce cell death in the host cells. Under a lethal dose MOI of 100, the resistance of EBL cells with knockout or knockdown of the NmrAL2_Bovine gene to cell death induced by *M. bovis* and *M. tb* was examined. The same number of NmrAL2-KO cells, NmrAL2_Bovine knockdown cells (si-NmrAL2), and WT EBL cells were seeded in 12-well plates. After 96 hours of infection with *M. bovis* and *M. tb*, the survival rate of NmrAL2-KO cells was significantly increased compared to the wild-type control group. Similarly, knockdown of NmrAL2_Bovine significantly inhibited cell death induced by *M. bovis* and *M. tb*. Figure 4 ).

[0034] Example 5. NmrAL2_Bovine gene inhibits intracellular NO production after Mycobacterium bovis infection.

[0035] With 3×10 per hole 5 Wild-type cell line EBL and knockout cell line NmrAL2-KO were seeded in 12-well plates at a ratio of 1:1 and cultured at 37°C and 5% CO2 until adherent. Before infection, the cells were washed three times with DMEM incomplete medium. *Mycobacterium bovis* (M. bovis) was used to infect both NmrAL2-KO and wild-type cell lines at an MOI of 10. Whole-cell lysates were collected 48 h after infection, and intracellular total NO concentration was measured using Griess Reagent. The results showed that the intracellular NO concentration in the NmrAL2_BovineKO cell line was significantly increased after 48 h of infection (p<0.01). Figure 5 The above results indicate that NmrAL2_Bovine inhibits intracellular NO production after Mycobacterium bovis infection.

Claims

1. Knockout NmrAL2_Bovine The use of a preparation of a gene in the manufacture of a medicament for the prevention or treatment of bovine tuberculosis, characterised in that, The formulation is a targeted knockout NmrAL2_Bovine a nucleic acid of the gene whose sequence is 5'-GGTATCTCGGTTGCTGATAT-3'.

2. knockdown NmrAL2_Bovine The use of the preparation of the gene in the preparation of a drug for preventing or treating bovine tuberculosis, characterized in that, The formulation is an siRNA targeting knockdown NmrAL2_Bovine of the gene with a targeting sequence of 5'-UGGCUUCUGCUAAGCUACCUCUGAG-3'.

3. A cell resistant to bovine tuberculosis, characterised in that, The endogenous NmrAL2_Bovine gene in the cell is knocked out or knocked down, so that the expression of the endogenous NmrAL2_Bovine gene is lost or reduced, the nucleic acid sequence of the knockout NmrAL2_Bovine gene is 5'-GGTATCTCGGTTGCTGATAT-3', the siRNA targeting sequence of the knockdown NmrAL2_Bovine gene is 5'-UGGCUUCUGCUAAGCUACCUCUGAG-3'.

4. The cell of claim 3, wherein, The preservation number of the cell is CCTCC NO: C2023350.

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