CN117777268A - Bovine cell mutants that knock out or silence the bovine rbmx2 gene and their application in resisting mycobacterial infections - Google Patents

Abstract

The invention discloses a construction method of a bovine rbmx2 gene knockout cell line based on a CRISPR-Cas9 gene editing technology, which adopts the CRISPR-Cas9 technology to knockout the rbmx2 gene sequence in EBL cells, establishes the EBL cell line of the rbmx2 gene knockout, confirms that the rbmx2 gene is a mycobacterium bovis infection dependent factor, determines that the EBL cell line of the rbmx2 gene knockout can resist mycobacterium bovis infection, and can be used for functional verification in human and livestock resisting mycobacterium bovis infection.

Description

Bovine cell mutant strain with knockout or silencing of bovine rbmx2 gene and its application in resisting mycobacterial infection

Technical field

The invention belongs to the biological field and relates to the application of bovine cell lines that knock out or silence the bovine rbmx2 gene in resisting mycobacterial infection.

Background technique

Bovine tuberculosis is a Class II animal disease in my country, mainly caused by Mycobacterium bovis. Mycobacterium bovis is also one of the main pathogens causing human tuberculosis. About 5-10% of human tuberculosis is caused by bovine tuberculosis. The fundamental reason for the lack of safe and efficient prevention and control measures for bovine and human tuberculosis is that the pathogenic mechanism of tuberculosis is not fully understood. In addition to macrophages and dendritic cells, lung epithelial cells are also one of the target cells of Mycobacterium tuberculosis. They play a role as a physical barrier and a biological barrier in the initial infection, and play a key role in activating macrophages. It plays a bridging role in killing and initiating acquired immunity. However, there are few reports on the mechanism of lung epithelial cells in the infection process of Mycobacterium tuberculosis, and the role of bovine lung epithelial cells in the infection process of Mycobacterium bovis is even closer to blank. Therefore, analyzing the role of bovine lung epithelial cells in Mycobacterium bovis infection is of great significance to analyzing the pathogenic mechanism of Mycobacterium bovis and discovering new target molecules against bovine tuberculosis, and can provide a reference for research on human tuberculosis.

RNA-binding proteins (RBPs) are an important class of proteins that bind to RNA and play a key role in post-transcriptional gene expression regulation. RBPs recognize and bind to specific mRNA conserved sequences through different RNA-binding domains. At least 1,500 RBPs have been discovered so far, and they are classified according to their different RNA-binding domains. RBPs are involved in regulating RNA metabolism in both the nucleus and cytoplasm. In the nucleus, RBPs are mainly involved in RNA processing, including splicing, polyadenylation, and nuclear export; in the cytoplasm, RBPs regulate mRNA localization, translation, and stability. Altered expression or abnormal function of RBPs can affect multiple steps of RNA metabolism, thereby inducing the occurrence of many types of diseases such as immune diseases, metabolic diseases, tumors, and neurological diseases.

As an RNA-binding protein, RBMX2 is mainly located in the nucleus. The encoded protein has an RNA recognition motif (RNA Recognition Motif, RRM) in its 56-134 amino acid residue region, and the function of this protein may be through the spliceosome. mRNA splicing, but as of now, the specific biological function of this protein is unknown, and its role in the pathogenesis of tuberculosis in humans and animals has not been reported.

Contents of the invention

The purpose of the present invention is to provide a gene editing technology for knocking out or silencing the bovine rbmx2 gene, and a fetal bovine lung cell line for knocking out or silencing the bovine rbmx2 gene, and to confirm its application in anti-mycobacterial infection, the Gene ID of the rbmx2 gene: 613705, or a gene encoding the protein shown in SEQ ID NO.1.

In order to achieve the above objects, the present invention adopts the following technical measures:

Knockout or silencing bovine rbmx2 gene technology and its application in creating cells resistant to mycobacteria (Mycobacteria) infection by knocking out or silencing bovine rbmx2 gene cell lines. Gene ID of the rbmx2 gene: 613705, or encoding SEQ ID The gene for the protein shown in NO.1.

The technology of knocking out or silencing the bovine rbmx2 gene and its application in creating cattle resistant to mycobacterial infection.

Application of preparations for knocking out or silencing bovine rbmx2 gene expression in the preparation of drugs against mycobacterial infection. The gene ID of the rbmx2 gene is: 613705, or the gene encoding the protein shown in SEQ ID NO.1.

Application of knocking out or silencing the bovine rbmx2 gene in preparing cell models resistant to mycobacterial infection.

In the above-mentioned applications, preferably, the knockout method is CRISPR/cas9 knockout technology, and the present invention can be achieved by not expressing RBMX2 protein in cells after knockout, or by losing or weakening the activity of RBMX2 protein;

In the above-mentioned applications, preferably, the selected knockout target is: GAATGAGCGTGAGGTCGAAC;

In the above application, preferably, the knocked-out cell contains the polynucleotide shown in SEQ ID NO.2;

In the above-mentioned applications, preferably, the mycobacteria include Mycobacterium bovis (Mycobacteriumbovis,

bovis), Mycobacterium smegmatis (M.s), and/or Bacillus Calmette guerin (BCG).

In the above-mentioned applications, preferably, the deposit number of the cell line in the cell model is CCTCC NO:

C2023298.

Compared with the prior art, the present invention has the following advantages:

1. The gene knockout cell line constructed in the present invention utilizes CRISPR-Cas9 gene editing technology and has the advantages of simple and fast construction method, low cost, and high mutation efficiency.

2. Since the gene knocked out is the rbmx2 gene, the gene knockout cell line constructed in the present invention can significantly inhibit the adhesion, invasion and invasion of Mycobacterium bovis (M.bovis) compared with wild-type EBL cells. Intracellular survival.

3. Since the gene knockout cell line constructed in the present invention is fetal bovine lung cells (EBL), it serves as the first biological and physical barrier against pathogens. Therefore, it can be used to effectively resist pathogenic invading cells and break through existing mycobacteria. The bottleneck problem of unknown disease resistance genes provides an important target for disease resistance breeding of Mycobacterium bovis.

Therefore, the present invention uses CRISPR-Cas9 technology to knock out the Mycobacterium bovis-dependent factor rbmx2 gene sequence in EBL cells and establish an EBL cell line with rbmx2 gene knockout, which can be effectively used for rbmx2 genes to resist Mycobacterium bovis infection in humans and animals. Functional verification in it has better application prospects in resisting bacterial invasion.

Description of the drawings

Figure 1: Schematic diagram of experimental results of electrophoresis detection of T7E1 enzyme digestion knockout efficiency;

The results show that there is efficient cleavage of sgRNA1.

Figure 2: Electrophoresis detection to identify the deletion results of RBMX2 monoclonal cell lines.

Figure 3: Schematic diagram of the sequencing results of RBMX2-KO#17 and RBMX2-KO#23 cell lines;

In the figure: WT: wild-type EBL cell sequence, - represents base deletion.

Figure 4: Relative quantitative PCR results of rbmx2 gene transcript level expression in RBMX2-KO#17 and RBMX2-KO#23. In the figure: WT is wild-type EBL, ***p<0.001, mean±SEM (n=3).

Figure 5: Verification results of proliferation phenotype of RBMX2-KO#17 and RBMX2-KO#23 cell lines;

In the figure: WT is wild-type EBL, *p<0.05, **p<0.01, ***p<0.001, mean±SEM (n=3).

Figure 6: Schematic diagram of the verification results of the death phenotype induced by Mycobacterium bovis infection in RBMX2-KO#17 and RBMX2-KO#23 cell lines;

In the figure: WT is wild-type EBL, *p<0.05, **p<0.01, ***p<0.001, mean±SEM (n=3).

Figure 7: Schematic diagram of verification results of RBMX2-KO#17 and RBMX2-KO#23 cell lines’ resistance to Mycobacterium bovis adhesion, invasion and intracellular survival phenotypes;

In the figure: WT is wild-type EBL, *p<0.05, **p<0.01, ***p<0.001, mean±SEM (n=3).

Figure 8: Schematic diagram of the verification results of the invasion phenotype of RBMX2-KO#17 cell line against Mycobacterium smegmatis and BCG;

In the figure: WT is wild-type EBL, *p<0.05, **p<0.01, ***p<0.001, mean±SEM (n=3).

Detailed ways

The technical solution of the present invention will be further described in detail below with reference to specific examples. It should be understood that the following examples are only used to explain the present invention but not to limit the scope of the present invention. Various modifications or equivalent substitutions made by those skilled in the art on the basis of the following embodiments should also be deemed to fall within the protection scope of the present invention. The experimental methods without specifying specific conditions in the following examples are usually implemented according to conventional conditions or the tool book "Molecular Cloning: Laboratory Guide" (NewYork: Cold Spring Harbor Laboratory, 1989), or according to the operation manual provided by the manufacturer. The suggested methods are implemented. Materials whose sources are not indicated in the examples, such as EBL cells (CN201910541509X), Cas9 protein, 293T cells, packaging plasmids, MSTN lentivirus, T vectors, etc., are all commonly used materials well known in the art and can be constructed by oneself according to literature reports or through Obtained through commercial means. Specific information on the plasmids used in this experiment can be found at

http://www.addgene.org/ query, Cas9 plasmid (#52962), restriction plasmid information is

pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP(#67991), packaging helper plasmid PMD2.G(#12259), PSPAX2(#12260).

Example 1: Construction of RBMX2 knockout EBL monoclonal cells

1. Construction and recovery of the bovine lung epithelial cell line (EBL) containing Cas9 protein EBL-Cas9

The construction of the EBL-Cas9 cell line is to integrate the LentiCas9 Blast plasmid into the EBL cells using a lentiviral delivery system, and then construct the EBL-Cas9 cell line through Baticidi drug screening. The lentiviral packaging in the lentiviral delivery system uses the "JetPRIME" reagent from PolyPlus, and the specific process is as follows;

(1) Take newly recovered HEK293T cells and culture them for about 3 generations for transfection. The day before transfection, cells were plated into a 10cm cell culture dish at 2*10 ⁶ per dish;

(2) Carry out transfection when the density of 293FT cells reaches 80%-90%. Before transfection, discard the old medium and add 5 mL of 5% FBSDMEM medium;

(3) Prepare transfection system. The total amount of transfection plasmid required for a 10cm cell culture m is 24ug (pMD2.G:psPAX2:LentiCas9 Blast plasmid = 1:2:3). Dilute the plasmid with 500uL JetPrime Buffer and vortex for 10 seconds to mix;

(4) Add 40uL of jetPRIMEreagent transfection reagent to the diluted plasmid and vortex for 1s to mix;

(5) After vortexing, the system will turn milky white. Let it stand at room temperature for 10 minutes. Then carefully add it to the cell culture medium and shake gently to mix;

(6) After 4-6 hours of transfection, the cells were replaced with medium. Discard the original culture medium and add 10 mL of 5% FBSDMEM culture medium;

(7) Cells were rehydrated 24 hours after cell medium replacement. Supplement 10mL of 5% FBSDMEM medium;

(8) About 66 hours after transfection, collect the cell supernatant in a 50ml centrifuge tube and centrifuge at 30000r/min, 4℃ for 10min;

(9) After centrifugation, take the supernatant and filter it into an ultracentrifuge tube with a 0.45um filter;

(10) Centrifuge at 30000r/min, 4℃ for 2.5h;

(11) After centrifugation, discard the supernatant, place it upside down on sterilized and dry filter paper to absorb the remaining liquid, then add 120uL pre-cooled sterile PBS and resuspend;

(12) Transfer the resuspended virus liquid to a 15 mL centrifuge tube, seal it with a parafilm and place it at 4°C for dispersion and dissolution overnight;

(13) After the virus is fully dissolved, it can be divided into aliquots according to needs and stored at -80°C to avoid repeated freezing and thawing.

Remove the EBL-Cas9 cell line from the liquid nitrogen and quickly place it in a 37°C water bath while shaking continuously to thaw the cells. Place the thawed cells into a sterile operating table, use a pipette to first draw 5 mL of cell culture medium (DMEM containing 5% FBS and 2% double antibody), then draw the cell suspension, pipet repeatedly several times, and pour into the centrifuge tube. Centrifuge at 1000r/min for 5 minutes and discard the supernatant. Add 2 mL of cell culture medium to a centrifuge tube, pipet repeatedly and evenly, then transfer the cells to a 6-well plate and culture them in a 37°C, 5% CO2 constant temperature incubator.

Subculture was performed when the cells covered approximately 95% to 100% of the 6-well plate. The specific steps are to take out the six-well plate covered with EBL-Cas9-I cells from the constant temperature incubator, discard the old culture medium, add 1 mL of PBS to wash, discard the PBS, add 1 mL of trypsin, and place at 37°C, 5% CO2 Incubate in a constant temperature incubator for 3 minutes, then observe whether the cells fall off, add an appropriate amount of cell culture medium and pipet repeatedly for more than 10 times, transfer to a 15mL centrifuge tube, centrifuge at 1000r/min for 5 minutes, and discard the supernatant. Add 6 mL of cell culture medium, mix by pipetting, transfer to 3 wells of a 6-well plate, and return to the incubator to continue culturing.

2. Design the sgRNA sequence targeting the RBMX2 gene and construct a recombinant plasmid

2.1 Design sgRNA sequence targeting RBMX2 gene

As shown in Table 1, an sgRNA was designed based on the sequence of RBMX2 (Gene ID: 613705) in NCBI, and a pair of primers was designed for plasmid construction based on the sgRNA.

Table 1 sgRNA sequence and plasmid construction primer sequence information

2.2 sgRNA plasmid construction

sgRNA annealing: Take 1 μL of the forward and reverse primers of sgRNA and 8 μL of ddH ₂ O as shown in Table 2, and perform the annealing procedure. That is: denaturation at 95°C for 10 minutes; annealing at 65°C for 60 minutes.

Table 2 sgRNA annealing system

Element System (μL) sgRNA-F 1 sgRNA-R 1 ddH ₂ O 8

Connect the target fragment to the T vector: Then prepare the reaction system according to Table 3 to perform the ligation reaction between the target fragment and the T vector (Company: Takara; Product No.: 6013). The program is 25°C for 30 minutes; 65°C for 10 minutes.

Table 3 T4 ligase reaction system

Element System (μL) T4 Buffer 2 restriction plasmid 4 insert DNA 10 T4 Ligase 2 ddH ₂ O 2 total 20

Transformation: Take 50 μL of competent cells melted in an ice bath (Company: Beijing Quanshijin Biotechnology; Product No.: CD201), add the above-mentioned ligation product, gently flick to mix, and place in an ice bath for 30 minutes; heat shock in a 42°C water bath for 45 seconds, and then quickly transfer the tube to ice for 2 minutes. Be careful not to shake the centrifuge tube during this process; add 500 μL of sterile LB culture medium to each centrifuge tube, mix well, and place in a shaker at 37°C, 200rpm for 1 hour to allow the bacteria to recover; then centrifuge at 4000rpm for 5 minutes, discard 350 μL of supernatant, and resuspend the transformed competent cells in the remaining 200 μL, add to the LB agar plate containing ampicillin resistance, spread the cells evenly, and after the liquid is absorbed, invert the plate and culture it at 37°C overnight.

Single clone detection: Pick a single clone and add it to LB liquid culture medium containing ampicillin resistance, place it on a shaker at 37°C and 200 rpm for expanded culture, take about 4-5 hours, and mix well. Take 200 μL of bacterial liquid and send it to Qingke for sequencing.

Extraction of sgRNA expression plasmid: Extract the sgRNA expression plasmid from the successfully sequenced single clone bacterial liquid according to the plasmid miniprep kit (Company: OmegaBio-Tek; Cat. No.: D6950-02).

3. Obtaining RBMX2 knockout EBL polyclonal cell line

Follow the method in step 1 of Example 1 to package the sgRNA expression plasmid with lentivirus, that is, replace the "LentiCas9 Blast plasmid" in the step with the sgRNA expression plasmid in step 2 above.

Then resuscitate EBL-Cas9 in a 6-well plate. After the cells are full, pass them at a ratio of 1:3. When the cells are full to about 60%, discard the old culture medium and add 1 mL of cell maintenance solution (containing 2% FBS+1) to each well. % double antibody in DMEM), then add 1 μL polybreme, and then add 40 μL sgRNA lentivirus respectively and mix well. After incubating for 24 hours, discard the old culture medium and add 2 mL of culture medium containing 5% FBS and 1% double antibody. After another 24 hours of incubation, replace 2 mL of DMEM culture medium containing 5% FBS, 1% double antibody and 2 μg/mL puromycin was used for drug screening.

After all the puromycin drug screen holes of the negative control (EBL-Cas9 cells without adding lentivirus) have died, the positive control and the negative control will be passaged (note: the positive control wells will be passaged into a 6-well plate at a ratio of 1:2, and the negative control wells will be passaged into 2 cells. Wells, one as a negative control drug screening group), so that a total of 7 days of drug screening were performed to obtain the RBMX2 knockout polyclonal EBL cell line. Some of these polyclonal cell lines were expanded and cultured for cryopreservation, and some were extracted using the TIANamp Genomic DNAKit kit to verify the knockout efficiency of the target site.

4. T7E1 enzyme digestion to verify knockout efficiency

According to the RBMX2 gene sequence, highly specific primers were designed near the knockout target site, and the amplification primer sequences are shown in Table 4.

Table 4 Primer sequence information for amplifying target fragments

Primer name Primer sequence information (5’-3’) sgRNA-F CCTTGCCCAATTTTTCGGAGG sgRNA-R ACAGGAGGATGGTAGTAACGG

Unify the concentration of the genome extracted in step 3 with a standard of 300ng/μL (±10ng/μL), and use the designed primers to perform PCR amplification. The PCR amplification reaction system is shown in Table 5.

Table 5 PCR amplification reaction system

Element System (μL) 2×preme STAR MAX premix 25 Primer-F 2.5 Primer-R 2.5 DNA Sample 0.5 ddH2O 19.5 total 50

The PCR reaction program was: pre-denaturation at 98°C for 5 min; denaturation at 98°C for 10 s, annealing at 57°C for 15 s, extension at 72°C for 30 s, 35 cycles; extension at 72°C for 5 min.

After determining the target band and size through 2% agar gel electrophoresis, use the Universal DNAPurification Kit to purify the PCR product and unify the product concentration to 20ng/μL (±2ng/μL), and perform a T7E1 enzyme digestion experiment. The reaction system is shown in Table 6.

Table 6 T7E1 enzyme digestion reaction system

The T7E1 enzyme digestion reaction procedure is as follows: denaturation at 95°C for 5 min without adding T7E1 enzyme; temperature decrease from 95°C to 85°C by 2°C/sec, and from 85°C to 25°C by 0.1°C/sec; adding 1 μL of T7E1 enzyme and mixing, and reacting at 37°C for 30 min.

The target band and size were determined again by 2% agar gel electrophoresis. The results are shown in Figure 1. The band brightness was used to calculate the knockout efficiency of the target, which proved that the knockout efficiency was above 10%, indicating that the target knockout efficiency designed in the present invention was high.

5. Selection of rbmx2 knockout EBL monoclonal cells

Resuscitate and culture the rbmx2 knockout EBL polyclonal cell line, and complete cell counting. Use the limiting dilution method to screen monoclonal cell lines, divide 100 cells into a 96-well plate at a rate of 1 to 2 cells/well, mark the culture wells containing only single cells, and observe them daily. After the monoclonal density is amplified to more than 80%, the cells are digested and transferred to a 6-well plate. Cell lines with good growth status are selected and the cell DNA is extracted using the TIANamp Genomic DNAKit kit. The rest are expanded and cultured for cryopreservation. Cell medium replacement: Use DMEM medium containing 10% FBS + 1% double antibody for cell medium replacement, with a time interval of 4 days.

PCR amplification of cellular DNA was performed, and the target band and size were determined by 2% agar gel electrophoresis again as shown in Figure 2. The product was sent to Qingke for sequencing. Through preliminary screening of mycobacterial infection phenotypes and combined with sequencing results, it was shown that two clones have the strongest resistance to mycobacterial infection, namely RBMX2-KO-#17 and RBMX2-KO-#23, indicating that although Knockout of this gene can improve the anti-mycobacterial ability of EBL cells, but due to the difference in frameshift mutations caused by the knockout, the anti-infection ability of the cell lines is different.

The sequencing results of the above two monoclonal clones are shown in Figure 3. Among them, RBMX2-KO-#17 was sent to the China Center for Type Culture Collection on October 24, 2023, and was classified and named: Bovine lung epithelial cells (Bovine lung epithelial cells) RBMX2-KO-#17, with a collection number of: CCTCC NO: C2023298, and the address is: Wuhan University, Wuhan, China.

The physiological and biochemical characteristics of the RBMX2-KO-#17 cell line are similar to those of the EBL fetal bovine lung epithelial cell line. The culture medium is DMEM high glucose + 10% FBS + 1% double antibody, pH 7.2-7.4, the culture temperature is 37°C, and 5% carbon dioxide is added.

Embodiment 2:

Verification of transcript levels in EBL cells with rbmx2 knockout

RNA was extracted using the Trizol method from the rbmx2 knockout monoclonal cell lines RBMX2-KO-#17, RBMX2-KO-#23 and the wild strain EBL, and reverse transcription kits were used (Company: Nanjing Novizan Biotechnology Co., Ltd. Co., Ltd.; Catalog No.: R223-01) reverse-transcribe RNA into cDNA, and then perform relative quantitative PCR verification. The primer sequences are as shown in Table 7, and the PCR reaction system is as shown in Table 8. The results show in Figure 4 that compared with wild-type EBL cells, the expression of the rbmx2 gene in the RBMX2 knockout monoclonal cell line was significantly reduced at the transcription level.

Table 7 Relative quantitative PCR primer sequence information

Primer name Primer sequence information (5’-3’) β-actin-F AGCAAGCAGGAGTACGATGAG β-actin-R ATCCAACCGACTGCTGTCA RBMX2-F TCCCACCGGCACAAAAAGTC RBMX2-R TCAGTGATACCTGAGCTCCC

Table 8 Relative quantitative PCR amplification reaction system

Element System(μL) 2×AceQ QPCR SYBR Green Master Mix 10 Primer-F 0.4 Primer-R 0.4 Template cDNA 2 ddH2O 7.2 total 20

The PCR reaction program is: pre-denaturation at 95°C for 5 minutes, cycle number is 1; denaturation at 95°C for 10 seconds, annealing at 60°C for 30 seconds, and the instrument default melting curve.

Example 3:

Verification of proliferation level of EBL cells with rbmx2 knockout

The wild-type EBL and the two rbmx2 knockout EBL cell lines numbered RBMX2-KO#17 and RBMX2-KO#23 were respectively revived in T25 cell flasks for subsequent experiments.

Cell proliferation test was performed by cck-8 staining. The logarithmic growth phase cells of the rbmx2 knockout monoclonal cell lines RBMX2-KO#17 and RBMX2-KO#23 and the wild strain were collected respectively. The cell suspension concentration was adjusted to 200ul. /well, the cell density to be tested was 20,000 cells/well, and the specifications were added to a 48-well plate, and the cells were cultured for 24 hours (5% CO2, 37°C). Then 20ul of CCK-8 solution was added to each well, incubated in the cell culture incubator for 4 hours, then taken out, and the absorbance of each well was measured at 450nm with a microplate reader. The results are shown in Figure 5. The results showed that rbmx2 knockout did not affect the proliferation rate and cell viability of EBL cells.

Embodiment 4:

Verification of the ability of rbmx2 knockout EBL cells to resist pathogens

Knockout cells RBMX2-KO#17, RBMX2-KO#23 and wild-type cells were seeded into a 48-well plate at 3 × 10 ⁴ per well, cultured for about 12 hours, and infected with Mycobacterium bovis (ATCC: 19210). (MOI=10), the experimental group and the uninfected control group were set up with 3 duplicate wells, and each cell was repeated 3 times. CCK-8 was used to detect cell survival rate 24h, 48h, 72h, and 96h after infection. The specific operation methods are as follows: (1) Discard the old culture medium in the cell wells and replace it with fresh culture medium; (2) Add fresh culture medium to the cell wells. Add 1/10 volume of CCK-8 solution; (3) Incubate the culture plate in the incubator for 1 hour; (4) Carefully pipet the supernatant into the enzyme plate without generating bubbles; (5) Use an enzyme reader to measure the wavelength at 450 nm The absorbance.

The results show that compared with the wild strain, the rbmx2 knockout monoclonal cell lines RBMX2-KO#17 and RBMX2-KO#23 can significantly improve the ability of EBL cells to resist Mycobacterium bovis infection, as shown in Figure 6.

Invasion assay: Knockout cells RBMX2-KO#17 and RBMX2-KO#23 and wild-type cells were seeded into 12-well plates at 2×10 ⁵ per well, and the invasion assay was started after about 12 h of culture. The cells were infected with Mycobacterium bovis at an MOI of 10 for 2 h, 4 h, and 6 h, followed by incubation with 100 μg/ml gentamicin for 2 h to kill extracellular bacteria. Subsequently, the infected cells were washed three times with phosphate-buffered saline (PBS) to eliminate extracellular bacteria. After sufficient washing, Triton X-100 (0.1%) was used to lyse the cells and plate counts were performed.

Adhesion test: Mycobacterium bovis with an MOI of 10 was used to infect RBMX2-KO#17, RBMX2-KO#23 and wild-type EBL cells respectively. The infection times were 15 minutes, 30 minutes, 1 hour and 2 hours respectively. Subsequently, the infected cells were washed three times with phosphate buffered saline (PBS) to eliminate extracellular bacteria. After extensive washing, cells were lysed with Triton X-100 (0.1%) and plate counted. The results show that bovine branch infection of rbmx2 knockout cell lines can significantly reduce their invasive ability.

Intracellular survival test: Mycobacterium bovis with an MOI of 10 was used to infect RBMX2-KO#17, RBMX2-KO#23 and wild-type EBL cells respectively, and then incubated with 100 μg/ml gentamicin for 2 hours to kill the cells. External bacteria. The infected cells were then washed three times with PBS to remove extracellular bacteria (referred to as 0h). Infected cells were lysed by adding Triton X-100 (0.1%) at 0, 24, 48 and 72 h after infection, and plate counting was performed.

The results are shown in Figure 7 (Figure 7 is the data of adhesion, invasion and intracellular survival from left to right). Compared with the wild strain, the rbmx2 knockout monoclonal cell lines RBMX2-KO#17 and RBMX2-KO#23 , can significantly reduce the adhesion and invasion of Mycobacterium bovis, reduce the survival of intracellular bacteria, and increase the level of cell resistance to pathogens.

According to the above invasion test steps, the monoclonal cell line RBMX2-KO#17 was investigated against Mycobacterium smegmatis (NC_008596.1, a gift from Professor Luiz Bermudez of Oregon State University) and Bacillus Calmette-Guérin (BCG) (ATCC: 35734, from Oregon State University). The anti-invasive ability of Professor Luiz Bermudez (gift from Professor Luiz Bermudez) (reference: Frontiers|Mycobacterium tuberculosis Rv0309 Dampens the Inflammatory Response and Enhances Mycobacterial Survival (frontiersin.org)), the results are shown in Figure 8, the rbmx2 knockout cell line can also significantly reduce smegmatis The invasive ability of mycobacteria and BCG.

Claims (8)

1. Knocking out or silencing rbmx2 gene of cow to create mycobacterium resisting agentMycobacteria) Use of the rbmx2 Gene in infected transgenic cattle, gene ID 613705, or Gene encoding the protein shown in SEQ ID NO. 1.

2. Use of a formulation for knocking out or silencing expression of a rbmx2 Gene of bovine in the preparation of a medicament against mycobacterial infection, said rbmx2 Gene having Gene ID 613705 or a Gene encoding a protein as set forth in SEQ ID No. 1.

3. Use of a rbmx2 gene knocked out or silenced in a bovine for preparing a cell model resistant to mycobacterial infection.

4. The use of claim 1 or 2 or 3 or 4, wherein the knockdown method is CRISPR/cas9 knockdown technique, wherein RBMX2 protein is not expressed in the cells after knockdown, or RBMX2 protein activity is lost.

5. The use of claim 4, wherein the CRISPR/cas9 knockout technique has selected knockout targets of: GAATGAGCGTGAGGTCGAAC.

6. The use according to claim 1 or 2 or 3 or 4, wherein the knocked-out cells comprise the polynucleotide shown in SEQ ID NO. 2.

7. The use according to claim 1, 2, 3 or 4, wherein the Mycobacterium is Mycobacterium bovisMycobacterium bovis, M.bovis) Mycobacterium smegmatis @Mycobacteria smegmatis, M.s) And/or BCG vaccineBacillus-calmette guerin, BCG)。

8. The use according to claim 3, wherein the cell line in the cell model has a preservation number of CCTCC NO: C2023298.