Application of NmrAL2Bovine gene as target spot in prevention and treatment of bovine tuberculosis

Abstract

The invention discloses an application of an NmrAL2Bovine gene as a target spot in prevention and treatment of bovine tuberculosis, the NmrAL2Bovine gene is knocked out or knocked down, so that the survival of mycobacterium bovis, mycobacterium tuberculosis, mycobacterium bovis BCG and mycobacterium smegmatis in host cells can be remarkably inhibited, and the normal proliferation of the host cells is not influenced. Therefore, an inhibitor of the NmrAL2Bovine gene can be used for preparing a medicine for preventing and treating bovine tuberculosis, and the NmrAL2Bovine gene can also be used as a target for designing transgenic cattle and is used for breeding or genetic therapy research for resisting bovine tuberculosis.

Description

Technical Field

[0001] The invention belongs to the technical field of animal disease resistance, and specifically relates to the application of NmrAL2_Bovine gene as a target in preventing and treating bovine tuberculosis. Background Art

[0002] Tuberculosis is an important zoonosis caused by infection with the Mycobacterium tuberculosis complex, which mainly includes Mycobacterium tuberculosis (M.tb) and its bovine variant Mycobacterium bovis (M.bovis). The World Organization for Animal Health (WOAH) has included animal tuberculosis in the list of diseases that need to be reported, and my country has listed bovine tuberculosis as a Class II animal disease and one of the eight key zoonoses. Since bovine tuberculosis is an important source of infection for human tuberculosis, it is of great significance in food safety and public health. All countries in the world have officially stipulated the use of a "quarantine-culling" strategy to control bovine tuberculosis. Developing countries are countries with a high burden of human and bovine tuberculosis. The "quarantine-culling" control of bovine tuberculosis has brought a heavy economic burden to governments and practitioners. At the same time, the huge burden of human tuberculosis and the increasing rate of bacterial resistance urgently require more effective prevention and control measures. Therefore, there is an urgent need in this field to develop a new method for effectively preventing and controlling zoonotic tuberculosis.

[0003] Nitric oxide (NO), as an important molecule, plays an important role in the regulation of tuberculosis. Moderate NO production can kill pathogenic microorganisms, while excessive NO production can lead to excessive inflammatory response, 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 NmrA-like protein widely distributed in higher animals, namely NMRAL1, also known as HSCARG. In the cytoplasm, NMRAL1 negatively regulates innate immunity by inhibiting the activity of NF-κB and RLR pathways. As a link between the cellular redox state and other signaling pathways, NMRAL1 inhibits the production of NO by binding to AS (Argininosuccinate synthetase, AS). ENSBTAG00000011305 is a gene of unknown function in cattle. Gene family analysis revealed that the gene contains the NmrA domain and belongs to the NmrA family protein, so we named the gene NmrAL2_Bovine. In general, the functions of NmrA family proteins in infectious diseases and immune regulation make it an important area for future research. However, to date, there has been no report on the role of NmrAL2_Bovine in the pathogenesis of bovine tuberculosis, and its mechanism of regulating mycobacterial infection needs further exploration. Summary of the invention

[0004] On the one hand, the present invention protects the use of the NmrAL2_Bovine gene inhibitor - for the preparation of a drug for preventing or treating bovine tuberculosis. Further, the NmrAL2_Bovine gene inhibitor includes a siRNA that targets and knocks down the expression of the NmrAL2_Bovine gene, or a nucleic acid molecule or its expression vector 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] On the one hand, the present invention protects a drug for preparing a drug for preventing or treating bovine tuberculosis, including a nucleic acid molecule or an expression vector thereof for targeted knockout of the NmrAL2_Bovine gene, preferably, the sequence of the nucleic acid molecule is 5'-GGTATCTCGGTTGCTGATAT-3'. It also includes a nucleic acid molecule for targeted knockdown of the NmrAL2_Bovine gene, i.e., a double-stranded RNA hybridized with the NmrAL2_Bovine gene, wherein the sequence of the first strand of the double-stranded RNA is the same as the target sequence in the NmrAL2_Bovine gene, and the target sequence is 5'-UGGCUUCUGCUAAGCUACCUCUGAG-3'.

[0006] On the other hand, the present invention protects a cell model against bovine tuberculosis, in which the endogenous Nm rAL2_Bovine gene in the cell model is knocked out or knocked down, thereby causing the expression of the endogenous NmrAL2_Bovine gene to be absent or reduced in expression. The successfully constructed NmrAL2_Bovine gene knockout cell line was named fetal bovine lung cell EBL / NmrA-KO#9 (abbreviated as NmrAL2-KO), which was deposited in the China Center for Type Culture Collection on November 14, 2023, with a deposit address of: Wuhan University, Wuhan, China, and a deposit number of CCTCC NO: C2023350.

[0007] On the other hand, the present invention protects a method for preparing a gene-edited cattle resistant to bovine tuberculosis, wherein the NmrAL2_Bovine gene in bovine cells is knocked out or knocked down, thereby causing the expression of the endogenous NmrAL2_Bovine gene to be absent or reduced in expression, and further preparing a gene-edited cattle with the NmrAL2_Bovine gene knocked out or knocked down.

[0008] The present invention also protects a method for screening candidate compounds for preventing and / or treating bovine tuberculosis, wherein a test group and a control group are set, wherein the test group is a group in which the test compound is added to the cells, and the control group is a group in which the test compound is not added to the cells, and the expression levels of the NmrAL2_Bovine gene in the test group and the control group are compared to screen the test compound that significantly reduces the expression level of the NmrAL2_Bovine gene.

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

[0010] 1. The present invention provides for the first time a new application of an 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. The present invention provides a genetically engineered cell resistant to infection by Mycobacterium tuberculosis complex and a method for preparing the same. The cell line can resist infection by Mycobacterium tuberculosis complex without affecting the normal function of the cells, and can be used as an in vitro experimental model to reduce the impact of chemical additives on cell experiments.

[0012] 3. The present invention provides a method for preparing gene-edited cattle resistant to bovine tuberculosis, and uses this method to prepare a bovine tuberculosis-resistant cattle model for use in the research of anti-tuberculosis drugs or preparations.

[0013] 4. The present invention provides a method for screening candidate compounds for preventing and / or treating tuberculosis, using the NmrAL2_Bovine gene as a target to screen test compounds that significantly reduce the expression level of the NmrAL2_Bovine gene. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0015] Figure 2 In Example 2, CCK8 was used to detect the effect of NmrAL2_Bovine gene knockout on EBL cell proliferation. WT-EBL is wild-type EBL cell, and NmrAL2-KO represents NmrAL2_Bovine gene knockout EBL cell. T test was used, ns, p>0.05.

[0016] Figure 3.In Example 3, CFU counts were used to detect the intracellular survival of bacteria after infection with M.bovis, M.tb, M.bovis BCG and Mycobacterium smegmatis in NmrAL2_Bovine knockout (AD) and knockdown expression (EH) cells. WT-EBL represents wild-type EBL cells, NmrAL2-KO represents NmrAL2_Bovine gene knockout cells constructed using CRISPR / Cas9 lentiviral strategy, si-NmrAL2 represents NmrAL2_Bovine gene knockdown expression cells constructed using RNAi technology, and NC siRNA is a 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, CCK8 was used to detect the cell survival rate of NmrAL2_Bovine knockout (A, B) and knockdown (C, D) cells infected with M.bovis and M.tb. ANOVA was used, ns, p>0.05; *, p<0.05; **, p<0.01; ***, p<0.001.

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

[0019] After extensive and in-depth research, the inventors discovered for the first time that editing the NmrAL2_Bovine gene inhibited the intracellular survival of M.bovis in host cells and reduced infection-induced host cell death. Using CRISPR / Cas9 technology, the NmrAL2_Bovine gene knockout cell line was constructed, and the NmrAL2_Bovine gene knockout cells were infected with M.bovis and M.tb strains. Through cell survival experiments and bacterial intracellular survival experiments, it was found that knocking out the NmrAL2_Bovine gene could significantly reduce the survival ability of M.bovis and M.tb in host cells and infection-induced host cell death, thus providing a new molecular target that can inhibit the intracellular survival of Mycobacterium tuberculosis complex.

[0020] The technical scheme of the present invention is further described in detail below in conjunction with specific examples. It should be understood that the following examples are only used to explain the present invention and are not used to limit the scope of protection of the present invention. Various modifications or equivalent substitutions made by those skilled in the art on the basis of the following examples should also be regarded as falling within the scope of protection of the present invention. The experimental methods for which specific conditions are not indicated in the following examples are usually implemented according to conventional conditions, or implemented according to the methods recommended in the operating manual provided by the manufacturer. Materials such as EBL cells, Cas9 proteins, HEK293FT cells, lentiviral packaging plasmid auxiliary plasmids, T vectors, etc., which are not indicated in the examples, are all commonly used materials known in the art and can be constructed according to literature reports or obtained through commercial channels. The specific information of the plasmids in this experiment can be found at http: / / www.addgene.org / , including Cas9 plasmids (#52962), restriction plasmids pKLV2-U6gRNA5 (BbsI)-PGKpuro2ABFP (#67991), packaging auxiliary plasmids pMD2.G (#12259), psPAX2 (#12260), etc.

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

[0022] 1.11 Design of specific sgRNA targeting NmrAL2_Bovine gene and construction of expression vector

[0023] Targeting the exon sequence of NmrAL2_Bovine gene (Ensembl: ENSBTAG00000011305), sgRNA was designed using sgRNAcas9 software (https: / / sourceforge.net / projects / sgrnacas9 / ), and then sgRNA targeting the exon of bovine NmrAL2_Bovine gene was selected based on the specificity evaluation results. The sequence of the selected sgRNA was: NmrAL2_Bovine-sgRNA: 5'-GGTATCTCGGTTGCTGATAT-3', where the PAM sequence for target recognition was "TGG". The sgRNA primer pairs sent to the company for synthesis were: NmrAL2_Bovine-sgR-F: 5'-CACCGGTATCTCGGTTGCTGATAT-3', NmrAL2_Bovine-sgR-R: 5'-AAACATATCAGCAACCGAGATACC-3'. Subsequently, 5 μL of the synthesized sgRNA primers F and R (10 pmol) were taken and annealed in a PCR instrument: 95°C, 10 min; 65°C, 60 min. Next, the annealed product was connected to the pKLV2-U6gRNA5 (BbsI)-PGKpuro2ABFP vector (Addgen #67991) linearized by BsmBI (NEB). Further, transformation, plating, and overnight culture were performed, and a single colony was picked for Sanger sequencing identification the next day. The positive clone colony was selected for expansion culture, and the plasmid was extracted using an endotoxin removal kit. The vector that obtained the correct expression of sgRNA was named pKLV2-U6gRNA5-NmrAL2_Bovine-KO.

[0024] 1.2 Preparation of NmrAL2_Bovine knockout cell line

[0025] HEK293FT cells were transfected with pMD2.G:psPAX2:pKLV2-U6gRNA5-NmrAL2_Bovine-KO at a mass ratio of 1:2:3 for lentiviral packaging. Subsequently, the bovine lung epithelial cell line EBL-Cas9 expressing a high-cleavage active Cas9 protein was inoculated with lentivirus (see CN118340888A for the method), and Puromycin drug screening was performed for 7 days. Then, the genomic DNA of the cells was extracted according to the method of the DNA extraction kit (KG203) of Tiangen Biochemical Technology (Beijing) Co., Ltd. Targeting the NmrAL2_Bov ine sgRNA target genomic region, NCBI-BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) was used to design the amplification primer pair NmrAL2_Bovine-PCR-F: 5'-AGCGACTCAGTGTTAC ATGGTG-3', NmrAL2_Bovine-PCR-R: 5'-TTCACCCCAACTCTGCTGTG-3'. Then, the genomic DNA was used as a template and the PCR reaction was performed using the amplification primer pair. Further, the PCR purified product was cloned into the pMD19-T vector, transformed, plated, and cultured overnight, and a single colony was picked for Sanger sequencing to identify the genotype of the monoclonal cell. The results showed that compared with wild-type cells, there was a 12-base deletion in the sgRNA target region in the genome of the NmrAL2-KO monoclonal cell ( Figure 1 A), indicating that a frameshift mutation occurred 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 the RT-qPCR method. Figure 1 As shown in Figure B, compared with wild-type cells, the transcription level of NmrAL2_Bovine in NmrAL2-KO knockout cells was significantly reduced, indicating that the NmrAL2_Bovine knockout cell line was successfully constructed and named fetal bovine lung cell EB L / NmrA-KO#9 (abbreviated as NmrAL2-KO). The cell line was deposited in the China Center for Type Culture Collection on November 14, 2023, with the deposit address: Wuhan University, Wuhan, China, and the deposit number CCTCC NO: C2023350.

[0026] Example 2. Knockout of the NmrAL2_Bovine gene does not affect normal cell proliferation

[0027] The CCK8 assay was used to evaluate the effect of NmrAL2_Bovine gene knockout on EBL cell proliferation. The experimental procedure was as follows: NmrAL2-KO cells were inoculated into 96-well culture plates, and CCK-8 was used to detect cell proliferation at 6, 12, 24, and 48 hours: (1) The old culture medium in the cell wells was discarded and replaced with fresh culture medium; (2) 1 / 10 volume of CCK-8 solution was added to the cell wells; (3) The culture plate was incubated in an incubator for 1 hour; (4) The supernatant was carefully pipetted into the ELISA plate without generating bubbles; (5) The absorbance at 450 nm was measured using an ELISA reader, and cell proliferation was calculated.

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

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

[0030] 3×10 per well 5 The wild-type cell line EBL and the knockout cell line NmrAL2-KO were inoculated in a 12-well cell plate at a ratio of 37°C and 5% CO2 until adherence, and washed 3 times with DMEM incomplete medium before infection. The mycobacteria used for infection (M.bovis, M.tb, M.bovis BCG and Mycobacterium smegmatis) were washed and dispersed with HBSS, and then inoculated in 12-well plates at an infection multiplicity (MOI) of 10:1 to make the bacteria and cells evenly contact. The 12-well plate was placed in a 5% CO2 incubator and incubated at 37°C for 6h. During this period, the initial infected bacterial solution was diluted 10 times continuously, and the appropriate dilution multiple was taken for colony counting. After the infection process was completed, the supernatant was discarded and washed 3 times with DMEM incomplete medium. 2mL DMEM incomplete medium (containing 2% FBS, 50μg / mL gentamicin) was added to each well and treated in the incubator for 1h to kill the residual bacteria outside the cell. After antibiotic treatment, wash with DMEM incomplete medium for 3 times, add 1mL DMEM medium (containing 2% FBS) to each well and continue culturing. This time is recorded as 0h after infection. Then, lyse the cells at different time points according to the experimental requirements, and take the lysate with appropriate dilution multiples for colony counting. Use a pipette to blow the lysed cell solution repeatedly to make it even, then take 100μL and dilute it 10 times continuously, take the lysate with appropriate dilution multiples and evenly spread it on the 7H11 plate, and count the colonies after culturing in a 37℃ incubator for about 3 weeks. Figure 3As can be seen in A, B, C, and D, the intracellular survival of M. bovis, M. tb, M. bovis BCG, and Mycobacterium smegmatis was significantly reduced in NmrAL2-KO compared with the wild-type control group.

[0031] Take EBL cells that have been passaged to 3-10 generations for the experiment, and wait for the cells to grow to 70%-80% for the transfection experiment. Prepare 1.5mL RNase-free EP tubes, add 150μL opti-MEM medium to each tube, add Lipofectamine 2000, and time for 5 minutes, which is recorded as tube A. Add the siRNA to be transfected to another tube, which is recorded as tube B. After 5 minutes, gently add the liquid in tube B drop by drop to tube A, and flick the bottom to mix. Let it stand at room temperature for 20 minutes to form an RNA-transfection reagent mixture (if the solution becomes turbid, it is a normal phenomenon and will not affect the transfection effect). Take out the cell culture plate and wash the cells to prevent the serum in the DMEM complete medium from affecting the transfection; wash it once with DMEM medium without serum, and then add opti-MEM medium. After 20 minutes, discard three wells and add three wells each time, and complete it in sequence. Slowly add the transfection mixture drop by drop to maximize the transfection efficiency. After transfection, the cell culture plate was placed in a cell culture incubator at 5% CO2 and 37°C. After 4-6 hours, the cells were washed once with serum-free DMEM medium, and then serum-free DMEM medium was added. After culturing for 18-24 hours, the cells were prepared for infection, and samples were taken at different time points after infection. In the experiment, the transfection concentrations of the negative control (NC siRNA) and the interfering RNA group siRNA were both 100nM. The sequence of the interfering siRNA targeting NmrAL2_Bovine was: 5'-UGGCUUCUGCUAAGCUACCUCUGAG-3', the synthesized sense chain was: 5'-UGGCUUCUGCUAAGCUACCUCUGAG-3', and the antisense chain was 5'-CUCAGAGGUAGCUUAGCAGAAGCCA-3'. Figure 1 As shown in C, the expression of NmrAL2_Bovine gene was successfully knocked down, and the constructed NmrAL2_Bovine gene knockdown expression cells were named si-NmrAL2. Further infection experiments proved that knocking down the expression of NmrAL2_Bovine gene could significantly reduce the intracellular survival of M.bovis, M.tb, M.bovis BCG and Mycobacterium smegmatis in EBL cells ( Figure 3 (E, F, G, H).

[0032] Example 4. Cells after knockout and knockdown of the NmrAL2_Bovine gene grow normally but are resistant to cell death induced by M.bovis and M.tb

[0033] After a certain dose of M.bovis and M.tb infect EBL cells, host cells will become pathological or even induced to die. Under the condition of lethal dose MOI=100, the resistance of EBL cells with knockout or knockdown of NmrAL2_Bovine gene to cell death induced by M.bovis and M.tb was detected. The same number of NmrAL2-KO cells, NmrAL2_Bovine gene knockdown cells si-NmrAL2 and WT EBL cells were first inoculated in 12-well plates. After 96h of infection with M.bovis and M.tb, it was found that the survival rate of NmrAL2-KO cells was significantly increased compared with the wild-type control group cells. Similarly, knockdown of NmrAL2_Bovine can significantly inhibit the cell death induced by M.bovis and M.tb ( Figure 4 ).

[0034] Example 5. NmrAL2_Bovine gene inhibits the production of NO in cells after Mycobacterium bovis infection

[0035] 3×10 per well 5 The wild-type cell line EBL and the knockout cell line NmrAL2-KO were inoculated in a 12-well cell plate at a ratio of 37°C and 5% CO2 until adherence. The cells were washed three times with DMEM incomplete medium before infection. Mycobacterium bovis (M.bovis) infected the NmrAL2-KO cell line and the wild-type cell line at MOI=10. After 48 hours of infection, the whole cell lysate was collected and the total intracellular NO concentration was detected using Griess Reagent. The results showed that the intracellular NO concentration of the NmrAL2_BovineKO cell line increased significantly after 48 hours of infection (p<0.01)( Figure 5 ). The above results showed that NmrAL2_Bovine inhibited the production of NO in cells after Mycobacterium bovis infection.

Claims

1. NmrAL2_Bovine Use of a gene inhibitor, characterized in that: Used for preparing medicines for preventing or treating bovine tuberculosis.

2. The use according to claim 1, characterized in that Said NmrAL2_Bovine Gene inhibitors including targeted knockdown NmrAL2_Bovine siRNA for gene expression, or targeted knockdown NmrAL2_Bovine A nucleic acid molecule of a gene or an expression vector thereof.

3. The use according to claim 2, characterized in that The targeting sequence of the knockdown siRNA is 5'-UGGCUUCUGCUAAGCUACCUCUGAG-3'.

4. The use according to claim 2, characterized in that The targeted knockout NmrAL2_Bovine The sequence of the nucleic acid molecule of the gene is 5'-GGTATCTCGGTTGCTGATAT-3'.

5. A drug for the prevention or treatment of bovine tuberculosis, characterized in that: Including targeted knockout or knockdown NmrAL2_ Bovine A nucleic acid molecule of a gene or an expression vector thereof.

6. The drug according to claim 5, characterized in that The targeted knockout NmrAL2_Bovine The sequence of the nucleic acid molecule of the gene is 5'-GGTATCTCGGTTGCTGATAT-3'.

7. The drug according to claim 5, characterized in that The targeted knockdown NmrAL2_Bovine The nucleic acid molecules of a gene include NmrAL2_Bovine A double-stranded RNA hybridized to a gene, wherein the sequence of the first strand of the double-stranded RNA is NmrAL2_ Bovine The target sequence in the gene is the same, and the target sequence is 5'-UGGCUUCUGCUAAGCUACCUCUGAG-3'.

8. A cell resistant to bovine tuberculosis, characterized in that Endogenous NmrAL2_Bovine The gene is knocked out or knocked down, so that the endogenous NmrAL2_Bovine Gene expression is absent or reduced.

9. The cell according to claim 8, characterized in that The deposit number of the cell is CCTCC NO: C2023350.

10. A method for preparing gene-edited cattle resistant to bovine tuberculosis, characterized in that: The steps include: NmrAL2 Gene knockout or knockdown, thereby making endogenous NmrAL2 The gene expression is missing or the expression level is reduced, and further preparation NmrAL2 Gene-edited cattle with gene knockout or knockdown.

11. A method for screening candidate compounds for preventing and / or treating bovine tuberculosis, characterized in that: include: A test group and a control group are set up, wherein the test group is a bovine cell with a test compound added thereto, and the control group is a bovine cell without a test compound added thereto, and the expression of the test compound in the cells of the test group and the control group is compared. NmrAL2_Bovine The expression level of the gene was significantly reduced by screening NmrAL2_Bovine Gene expression levels of test compounds.

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