Use of biological products for silencing or knocking out id2 gene in regulating host's anti-brucella infection

Abstract

This invention belongs to the field of zoonotic disease prevention and control and molecular immunology technology, specifically relating to the application of a biological product that silences or knocks out the ID2 gene in regulating host resistance to Brucella infection. This invention constructs an ID2 gene knockout macrophage model using CRISPR / Cas9 gene editing technology and establishes a Brucella infection system. Experiments show that ID2 gene knockout significantly enhances the host's ability to clear Brucella. RNA-seq analysis shows that ID2 participates in regulating the host's transcriptional response network to Brucella, with its differentially expressed genes significantly enriched in lipid metabolism and autophagy. Mechanistic results indicate that the ID2 gene participates in the regulation of Brucella-mediated lipophage. It can be seen that the ID2 gene affects the intracellular replication level and lipophage process of Brucella. This invention provides new targets and theoretical basis for drug development against Brucella infection, immune regulation, and disease-resistant breeding.

Description

Technical Field

[0001] This invention belongs to the field of zoonotic disease prevention and control and molecular immunology technology, specifically relating to the application of a biological product that silences or knocks out the ID2 gene in regulating the host's resistance to Brucella infection. Background Technology

[0002] Brucella ( Brucella spp. Brucella is a typical zoonotic pathogen, and the brucellosis it causes has long threatened animal health and public health safety.

[0003] The World Health Organization's (WHO) "One Health" concept and its "human disease prevention in animals, and prevention at the source" strategy both emphasize the forward-looking and systematic nature of zoonotic pathogen prevention and control. Against this backdrop, systematically analyzing the interaction mechanisms between Brucella and the host immune system, and identifying key host factors regulating the infection process, has become a current research hotspot and strategic breakthrough.

[0004] Differentiation repressor 2 (ID2, also known as DNA-binding repressor protein 2) is a member of the ID protein family and plays a crucial role in cell differentiation and development. Previous studies have shown that knocking out ID2 in RAW264.7 cells can enhance the innate immune response, thereby inhibiting SARS-CoV-2 infection. Therefore, further research is needed to expand the application of the ID2 gene. Summary of the Invention

[0005] To address the aforementioned issues, this invention provides an application of a biological product that silences or knocks out the ID2 gene in regulating host resistance to Brucella infection. The specific technical solution is as follows.

[0006] The application of biological products that silence or knock out the ID2 gene in regulating host resistance to Brucella infection, wherein the NCBI accession number of the ID2 gene is Gene ID: 15902, and the nucleotide sequence of the coding region of the ID2 gene is shown in SEQ ID NO.1; The amino acid sequence encoded by the ID2 gene is shown in SEQ ID NO.2; The biological product is used to knock out or silence the ID2 gene to regulate the host's resistance to Brucella infection.

[0007] In another preferred embodiment, the regulation of host resistance to Brucella infection refers to enhancing the host's resistance to Brucella infection and inhibiting the proliferation of Brucella in host cells.

[0008] In another preferred embodiment, the biological product comprises a reagent for knocking out or silencing the ID2 gene. This invention demonstrated that knocking out the ID2 gene in RAW264.7 cells using CRISPR / Cas9 technology significantly reduced the intracellular survival and replication levels of Brucella. Therefore, gene-editing reagents for knocking out or silencing the ID2 gene can be used to develop drugs against Brucella infection or to construct anti-infection animal models.

[0009] In another preferred embodiment, the reagent includes the Cas9 gene-editing protein or its expression vector, and the sgRNA or its expression vector that guides Cas9 to specifically recognize the ID2 gene.

[0010] It should be noted that, in addition to the CRISPR / Cas9 system, any technical means that can achieve ID2 gene knockout or expression inhibition, including RNA interference (RNAi), CRISPRi, and antisense oligonucleotide technology, are all within the scope of protection of this invention.

[0011] In another preferred embodiment, the sgRNA gene has two copies, namely sgRNA-1 and sgRNA-2; The nucleotide sequence of the sgRNA-1 is shown in SEQ ID NO:3; The nucleotide sequence of the sgRNA-2 is shown in SEQ ID NO:4.

[0012] In another preferred embodiment, the expression vector for the sgRNA is a vector in which the sgRNA gene is inserted into pGL3-Lentin-U6-sgRNA-EF-1α promoter-Puromycin. BsmB I site was obtained.

[0013] In another preferred embodiment, the biological product further includes a RAW264.7 cell line resistant to Brucella infection; The RAW264.7 cell line resistant to Brucella infection was obtained by knocking out or silencing the ID2 gene in the cell line. This RAW264.7 cell line resistant to Brucella infection exhibits a significant ability to inhibit Brucella proliferation and can be used for Brucella infection mechanism research, drug screening, and host factor function verification.

[0014] Specifically, the process for obtaining the RAW264.7 cell line resistant to Brucella infection is as follows: After the expression vector of the sgRNA is packaged and screened by lentivirus, it is used to infect host cells expressing Cas9 protein to obtain ID2 gene knockout polyclonal cells. Stable monoclonal cell lines are obtained through screening, which are the RAW264.7 cell lines resistant to Brucella infection.

[0015] In another preferred embodiment, Brucella is introduced into a Brucella-resistant RAW264.7 cell line to modulate the host's resistance to Brucella infection.

[0016] In another preferred embodiment, the Brucella is Brucella-M5.

[0017] In another preferred embodiment, the amino acid sequence of the protein encoded by the ID2 gene has a protein accession number of P41136 in the UniProt database.

[0018] Compared with the prior art, the present invention has the following beneficial effects: This invention utilizes CRISPR / Cas9 gene editing technology to construct an sgRNA vector targeting the ID2 gene in RAW264.7 cells. After lentiviral packaging and screening, an ID2 gene knockout macrophage cell line and a Brucella infection model were successfully established. Bacteriological results showed that, compared with wild-type cells, ID2 gene knockout cells exhibited significant resistance to Brucella infection, with a significantly reduced intracellular bacterial load, indicating that ID2 promotes Brucella proliferation in host cells. Furthermore, in the in vitro infection system, the ID2 gene positively regulates Brucella replication within host cells. Brucella infection induces lipophage in host cells, and ID2 gene knockout significantly reduced the co-localization level of lipid droplets and the lysosomal marker LAMP1 (by approximately 6.73%), suggesting that ID2 participates in regulating the lipophage process. Simultaneously, both drug inhibition and gene intervention methods to inhibit lipophage significantly suppressed Brucella proliferation within host cells. Attached Figure Description

[0019] Figure 1 The first graph shows the results of ID2 promoting Brucella infection in RAW264.7 cells; A and B show the results of Western blotting detection of ID2 protein expression levels in RAW264.7 cells infected with Brucella at 0h, 12h, 24h, and 48h, with B being a bar chart of A; C shows the nucleic acid sequence comparison between ID2 gene knockout monoclonal cell lines and wild-type (WT) cells, with sgRNA target sites marked in red and PAM sequences marked in blue; D shows the results of viability detection of ID2 knockout cells and blank control group cells using the CCK-8 assay; E shows the verification of ID2 knockout effect in RAW264.7 cells using Western blotting; F shows the results of fluorescence detection after collecting cells from the control group and ID2 knockout group cells infected with Brucella at 24h, 48h, and 72h; G shows the results of colony forming unit (CFU) count detection after infecting control group and ID2 knockout group cells with Brucella at 24h, 48h, and 72h.

[0020] Figure 2 Figure A shows the results of transcriptome sequencing experimental data analysis; Figure B shows the volcano diagram of differentially expressed genes (DEGs) identified by RNA-seq between the blank control group and the ID2-KO cell group; Figure C shows the KEGG pathway enrichment analysis of differentially expressed genes between the blank control group and the ID2-KO cell group after Brucella infection; Figures C and D show the gene set enrichment analysis of ID2-regulated genes.

[0021] Figure 3 Figure 1 shows the results of ID2's involvement in lipid autophagy. Figures A-E show the protein levels of LC3I, LC3II, p62, Becn1, and PLIN2 in the blank control group and ID2 knockout cells after 0h, 12h, 24h, 48h, and 72h of Brucella infection. Figures B-E show the corresponding bar charts in A. Figure F shows the results of confocal microscopy observation of cells treated with OA in the control group, ID2 knockout cells, and NF-κB activator 2. Figure G shows the results of quantitative analysis of the proportion of colocalized spots in the control group, ID2 knockout cells, and NF-κB activator 2 treated with OA. Scale bar: 10μm. Figure H shows the results of triglyceride detection in the control group and ID2 knockout cells 24 hours after Brucella infection. Figure I shows the results of total cholesterol (TC) detection in the control group and ID2 knockout cells 24 hours after Brucella infection. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0023] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] The room temperature in the following examples is 37°C.

[0025] Example 1: Construction of ID2 gene knockout cell line The coding region nucleotide sequence of the ID2 gene is shown in SEQ ID NO.1; SEQ ID NO.1: ATGAAAGCCTTCAGTCCGGTGAGGTCCGTTAGGAAAAACAGCCTGTCGGACCACAGCTTGGGCATCTCCCGGAGCAAAACCCCGGTGGACGACCCGATGAGTCTGCTCTACAACATGAACGACTGCTACTCCAAGCTCAAGGAACTGGTGCCCAGCATCCCCCAGAACAAGAAGGTGACCAAGATGGAAATCCTGCAGCA CGTCATCGATTACATCTTGGACCTGCAGATCGCCCTGGACTCGCATCCCACTATCGTCAGCCTGCATCACCAGAGACCTGGACAGAACCAGGCGTCCAGGACGCCGCTGACCCTGAACACGGACATCAGCATCCTGTCCTTGCAGGCATCTGAATTCCCTTCTGAGCTTATGTCGAATGATAGCAAAGTACTCTGTGGCTAA.

[0026] The amino acid sequence encoded by the ID2 gene is shown in SEQ ID NO.2; SEQ ID NO. 2: MKAFSPVRSVRKNSLSDHSLGISRSKTPVDDPMSLLYNMNDCYSKLKELVPSIPQNKKVTKMEILQHVIDYILDLQIALDSHPTIVSLHHQRPGQNQASRTPLTTLNTDISILSLQASEFPSELMSNDSKVLCG.

[0027] A mouse macrophage cell line with ID2 gene knockout RAW264.7 was constructed using CRISPR / Cas9 technology, and monoclonal cells were obtained through screening.

[0028] 1. Plasmid vector construction The Lentin-U6-sgRNA cell expression vector was constructed using pGL3-Lentin-U6-sgRNA-EF-1αpromoter-Puromycin as the basic backbone. Two complementary single-stranded sgRNAs were annealed to form a double strand with sticky ends. After being ligated into the backbone, single clones were selected for routine Sanger sequencing identification, and then plasmid vectors were mass-produced. The specific construction method is as follows:

[0029] (1) Design sgRNA sequences Download the target sequence from the NCBI database, select -NGG near its 3' end as the PAM site for the sgRNA, and ensure that the target mutation site is located in the first 50% of the protein sequence from the N end.

[0030] Primer sequence design referenced Zhang Feng's sgRNA design website: https: / / zlab.squarespace.com / guide-design-resources.

[0031] (2) Constructing a linearized skeleton carrier The pGL3-Lentin-U6-sgRNA-EF-1α promoter-Puromycin backbone vector contains sites at +406bp and +2291bp that can be activated by… BsmB The specific sites recognized by the I-endonuclease yield linearized vectors with double sticky ends after digestion. The plasmid digestion reaction system is shown in Table 1.

[0032] Table 1 Plasmid Enzyme Digestion Reaction System Transfer the prepared components to centrifuge tubes and incubate at 56°C for 2 hours. Identify the enzyme digestion products by 1.2% agarose gel electrophoresis, and recover them using a gel extraction purification kit. After determining the concentration, store at -20°C for later use.

[0033] (3) Synthesis of sgRNA double-stranded DNA To obtain a double-stranded sgRNA fragment compatible with the ends of a linearized plasmid vector, oligonucleotide primers containing sticky ends were first designed and synthesized by a biotechnology company. Specifically, a CACCG sticky adapter sequence was added to the 5' end of the upstream primer, and AAAC and C sticky adapter sequences were added to the 5' and 3' ends of the downstream primer, respectively, to ensure accurate pairing with the corresponding sticky ends of the backbone vector.

[0034] The synthesized upstream and downstream oligonucleotides were delivered as lyophilized powders, centrifuged at 10,000 rpm for 3 min to thoroughly mix, and then diluted separately to 100 μM for later use. Annealing was then performed to form a double-stranded structure. The primer mixture was run in a PCR instrument with the following temperature control programs sequentially: 95℃, 5 min; 95℃~85℃, -2℃ / cycle, 10 cycles; 85℃ to 25℃, -0.1℃ / cycle, 600 cycles; 4℃, forever. The target gene sgRNA sequence is shown in Table 2.

[0035] Table 2. Target gene sgRNA sequence (4) Carrier connection The linearized backbone vector was ligated with the annealed sgRNA double-stranded oligonucleotides. Full-grain gold T4 DNA ligase was added to the reaction system, and the ligation mixture was prepared according to the manufacturer's recommended ratio. The reaction solution was incubated in a 25°C water bath for 15 min to complete the ligation between the insert and the vector. The plasmid ligation reaction system is shown in Table 3.

[0036] Table 3 Plasmid ligation reaction system (5) Sequencing identification and preparation of monoclonal vectors The ligation product was transformed into competent DH5α Escherichia coli cells and incubated overnight at 37°C. Single colonies were then picked from the plate and inoculated into LB broth containing ampicillin for amplification. The obtained bacterial culture was then sent to a third-party company for Sanger sequencing verification after plasmid extraction.

[0037] After sequencing confirmed the accuracy of the target sgRNA sequence, a high-purity plasmid was extracted using an endotoxin-removing plasmid extraction kit, and its concentration was determined using a micro spectrophotometer. The qualified sgRNA plasmid vector was stored at −20℃ for subsequent experiments.

[0038] 2. Lentiviral Packaging and Cell Infection (1) One day before transfection, HEK293T cells were evenly spread in a culture dish to ensure that the cell confluence was maintained between 70% and 80% the day before transfection.

[0039] (2) On the day of transfection, discard the original culture medium and replace it with fresh DMEM medium containing 2% fetal bovine serum and no antibiotics to improve the virus packaging efficiency and obtain cell culture medium.

[0040] (3) According to the transfection system requirements, use 500 μL JetPrime Buffer to dilute the sgRNA plasmid vector DNA to be transfected, mix thoroughly and then centrifuge briefly.

[0041] (4) Add 40 μL of JetPrime transfection reagent to the above sgRNA plasmid vector DNA mixture, vortex gently for 10 s, centrifuge briefly, and let stand at room temperature for 10 min to allow the complex to form. Then slowly add it dropwise to the cell culture medium, gently shake to avoid excessive local concentration.

[0042] (5) 24 h after transfection, the culture medium was replaced with 10 mL of DMEM medium containing 2% FBS and antibiotics (double anti-antibody) to inhibit contamination and maintain cell status.

[0043] (6) 48 h after transfection, the culture supernatant was collected, centrifuged at 3500 g for 10 min to remove cell debris and obtain the packaged virus; the clear supernatant was transferred into sterile cryovials and stored at −80℃ for subsequent infection experiments. The lentivirus packaging plasmid transfection system is shown in Table 4.

[0044] Table 4 Lentiviral Packaging Plasmid Transfection System 3. Titer determination (1) The day before infection, RAW264.7 cells were seeded into six-well plates to ensure that the cell confluence reached about 50% the next day.

[0045] (2) The packaged virus was diluted in DMEM culture medium containing 1% double antibody and 3% FBS at different volumes (10μL, 20μL, 50μL, 100μL, 200μL) to set up a control group.

[0046] (3) After 24 hours of infection, replace the medium with fresh medium containing 1% antibiotics and 3% FBS and continue incubation.

[0047] (4) After 72 hours of infection, puromycin was added for screening. The treatment was continued for 3 days, and then DMEM medium was used for further culture to obtain monoclonal cells.

[0048] 4. Identification of knockout cells When the monoclonal cells were passaged into 6-well plates, a portion of the cells were collected to extract the genome. The target region was amplified using Novizan P515 high-fidelity polymerase, and Sanger sequencing was performed to identify the gene editing status. Finally, the ID2 gene knockout RAW264.7 cell line was obtained and designated as ID2-KO cells.

[0049] Example 2: ID2 gene knockout inhibits Brucella replication 1. Brucella infection experiment Brucella M5 strain was provided by the Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences. The specific infection process of Brucella infecting RAW264.7 cells is as follows: 2×10 one day in advance 6ID2-KO cells were seeded in 6-well plates and cultured in DMEM medium containing 2% FBS. Brucella bacterial suspension was prepared one day in advance, centrifuged at 5000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in DMEM. The suspension was added to the cell plate at an MOI of 100, gently mixed, centrifuged at 400g for 10 min, carefully transferred to an incubator, and incubated statically for 1 h. After challenge for 1 h, the cells were washed three times with PBS, and cultured in 2% FBS + 25 μg / mL gentamicin medium for further incubation to obtain the ID2-KO cell group. RAW264.7 cells infected with Brucella without ID2 gene knockout (wild-type cells) served as the blank control group.

[0050] 2. Brucella infection promotes ID2 protein expression levels like Figure 1 As shown in Figures A and B, the ID2 protein level was significantly increased in Brucella-infected cells compared to the blank control group. The specific steps for Western blotting detection of the protein were as follows: After extracting total intracellular protein, SDS-PAGE electrophoresis was performed at a constant voltage of 80V. Once the sample migrated to the separating gel interface, the voltage was switched to 100V and continued until the bromophenol blue front was near the bottom of the gel. Subsequently, the membrane was transferred at a constant current of 250mA for 120 min. After transfer, the membrane was transferred to blocking buffer and incubated at room temperature with shaking for 2 h. After blocking, the membrane was transferred to diluted primary antibody solution and incubated overnight at 4°C. The next day, the membrane was washed three times with PBST buffer for 10 min each time. Then, it was transferred to HRP-labeled secondary antibody working solution and incubated on a shaker at room temperature for 2 h. After the secondary antibody reaction, the membrane surface was treated with an ECL chemiluminescence substrate kit, and after exposure, the target protein bands were captured and images recorded using a chemiluminescence imaging analyzer.

[0051] The viability of ID2-KO cells was compared with that of the blank control group using the CCK-8 assay. The results are as follows: Figure 1 As shown in D in the figure, ID2 expression in ID2-KO cells is significantly reduced, while cell viability remains unchanged. The specific detection steps are as follows: ID2-KO cells in logarithmic growth phase and blank control cells were digested, counted, and seeded into 96-well plates at the designed density. Cells were cultured for 3 days, and cell viability was measured at days 0, 1, 2, and 3. 10 μL of CCK8 solution was added to each well, gently mixed, and the plates were returned to the incubator for 4 hours in the dark. The absorbance of each well was measured at 450 nm using a microplate reader.

[0052] 3. ID2 gene knockout inhibited Brucella infection. To further investigate the Brucella bacterial load in ID2 knockout cells, normal RAW264.7 cells were infected with Brucella (control group) and ID2-KO cells (ID2 knockout group). Cell counts and fluorescence detection were performed at 24, 48, and 72 hours. The specific procedure for cell counts was as follows: cells were challenged with Brucella M5 strain (MOI=100) for 1 h, washed three times with PBS, and cultured in medium containing 25 μg / mL gentamicin to inhibit extracellular bacterial growth. Cells were then cultured for 24, 48, and 72 h, lysed, serially diluted, and plated. The number of CFU per well was calculated. The specific procedure for fluorescence detection was as follows: after plating the ID2 knockout group and control group, cells were infected with red fluorescent Brucella for 24, 48, and 72 h. Cells were washed twice with PBS, treated with DAPI nuclear staining solution for 20 min, discarded, and the level of Brucella in the cells was observed and photographed under a fluorescence microscope.

[0053] After collecting the cells, fluorescence detection was performed separately. The results are as follows: Figure 1 As shown in F, compared with the control group cells, the ID2 gene knockout group cells showed a weaker red fluorescent protein (RFP) signal, indicating that ID2 knockout can inhibit Brucella replication. Colony forming unit (CFU) count results are shown in... Figure 1 As shown in G, the number of CFUs in the IID2 gene knockout group cells was significantly lower than that in the control group cells.

[0054] In summary, the data above indicate that Brucella infection can upregulate ID2 expression, and the ID2 gene is crucial for efficient Brucella replication in RAW264.7 cells.

[0055] Example 3: The ID2 gene affects the regulation of lipid synthesis and autophagy in Brucella. 1. RNA sequencing analysis To perform RNA transcriptome analysis, RNA sequencing libraries were constructed from the ID2-KO cell group and the blank control group. All samples were infected with Brucella at MOI=100, and cell samples were collected 24 hours post-infection. Each sample was tested in triplicate. Transcriptome sequencing and analysis were performed by BGI Genomics using DNBSEQ technology. A corrected p-value ≤0.05 and an absolute fold change ≥1 were set as the threshold for significant differential expression. Subsequently, differentially expressed genes (DEGs) in different groups were analyzed using the BGI Genomics platform and Gene Set Enrichment Analysis (GSEA). The results are as follows: Figure 2 As shown, from Figure 2 As shown in Figure A, a total of 2437 upregulated genes and 2316 downregulated genes were identified in the ID2-KO cell group. From... Figure 2As shown in B, KEGG pathway enrichment analysis revealed a significant upregulation of autophagy-related gene expression. Figure 2 As shown in C and D, gene set enrichment analysis (GSEA) revealed a significant enrichment of fatty acid and cholesterol biosynthesis pathways in the ID2-KO cell group. These results suggest that ID2 knockout can induce lipid synthesis and autophagy.

[0056] Example 4: ID2 gene participates in cellular lipophage process 1. Cell challenge treatment The ID2-KO cell group and the blank control group were infected with Brucella at 0, 12, 24, 48 and 72 hours, respectively. The specific infection steps are as described in step 1 of Example 2.

[0057] 2. Validation of key lipophage protein levels in ID2 gene knockout cells Protein levels of LC3I, LC3II, p62, Becn1, and PLIN2 were assessed using Western blot analysis. The specific steps are described in step 2 of Example 2.

[0058] Figure 3 As shown in A~E, after Brucella infection, the protein levels of BECN1 and LC3B-II in the ID2-KO cell group were significantly reduced; at the same time, compared with the blank control group, the protein level of PLIN2 in ID2-KO cells was significantly increased.

[0059] 3. Confocal validation of lipophage levels in ID2 gene knockout cells RAW264.7 cells in the MOCK group (without ID2 gene knockout), the DMSO control group (RAW264.7 cells that did not undergo ID2 gene knockout but received the same volume of DMSO treatment as the ID2-KO cell group), and the ID2-KO cell group were all treated with oleic acid and then observed using a confocal microscope. The specific procedure is as follows: The cells were fixed with 4% paraformaldehyde for 30 minutes at room temperature. They were then permeabilized with Triton for 10 minutes. Next, the cells were blocked with 5% FBS for 1 hour at room temperature. After permeabilization and blocking, the cells were incubated with Lamp1 antibody. Subsequently, the cells were stained with BODIPY 493 / 503. The cell nuclei were stained with DAPI. The results are as follows: Figure 3 As shown in F and G in the figure, it can be seen from the figure that the knockout of the ID2 gene resulted in a 6.73% reduction in the colocalization signal between LAMP1 and lipid droplets.

[0060] 4. Detection of cholesterol and triglyceride levels in ID2 gene knockout cells All three groups of cells were treated with 1.2 × 10⁶ cells / day. 6 Cells were seeded at a density of [number] cells / well in 6-well plates and cultured for 24 hours. Cell lysis buffer was extracted using lysis buffer, and the cells were centrifuged at 2000 rpm for 5 minutes at room temperature. The supernatant was collected. Triglyceride and total cholesterol levels were determined using a biochemical assay kit according to the kit instructions.

[0061] Figure 3 The H and I values ​​indicate that knocking out ID2 leads to a decrease in intracellular total cholesterol and triglyceride levels, suggesting that ID2 may play a role in lipid synthesis.

[0062] In summary, ID2 positively regulates cellular lipoplasmosis during Brucella infection.

[0063] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of biological products that silence or knock out the ID2 gene in regulating host resistance to Brucella infection, characterized in that, The NCBI accession number for the ID2 gene is Gene ID: 15902, and the nucleotide sequence of the coding region of the ID2 gene is shown in SEQ ID NO. 1; The amino acid sequence encoded by the ID2 gene is shown in SEQ ID NO.2; The biological product is used to knock out or silence the ID2 gene to regulate the host's resistance to Brucella infection.

2. The application of the biological product for silencing or knocking out the ID2 gene according to claim 1 in regulating host resistance to Brucella infection, characterized in that, The regulation of host resistance to Brucella infection refers to enhancing the host's resistance to Brucella infection and inhibiting the proliferation of Brucella in host cells.

3. The application of the biological product for silencing or knocking out the ID2 gene according to claim 1 in regulating host resistance to Brucella infection, characterized in that, The biological products include reagents for knocking out or silencing the ID2 gene.

4. The application of the biological product for silencing or knocking out the ID2 gene according to claim 3 in regulating host resistance to Brucella infection, characterized in that, The reagents include the Cas9 gene-editing protein or its expression vector, and the sgRNA or its expression vector that guides Cas9 to specifically recognize the ID2 gene.

5. The application of the biological product for silencing or knocking out the ID2 gene according to claim 4 in regulating host resistance to Brucella infection, characterized in that, The sgRNA gene has two genes, namely sgRNA-1 and sgRNA-2; The nucleotide sequence of the sgRNA-1 is shown in SEQ ID NO:3; The nucleotide sequence of the sgRNA-2 is shown in SEQ ID NO:

4.

6. The application of the biological product that silences or knocks out the ID2 gene according to claim 5 in regulating host resistance to Brucella infection, characterized in that, The expression vector for the sgRNA is formed by inserting the sgRNA gene into pGL3-Lentin-U6-sgRNA-EF-1α promoter-Puromycin. BsmB I site was obtained.

7. The application of the biological product for silencing or knocking out the ID2 gene according to claim 5 in regulating host resistance to Brucella infection, characterized in that, The biological product also includes the RAW264.7 cell line, which is resistant to Brucella infection; The RAW264.7 cell line resistant to Brucella infection was obtained by knocking out or silencing the ID2 gene in the cell line.

8. The application of the biological product that silences or knocks out the ID2 gene according to claim 7 in regulating host resistance to Brucella infection, characterized in that, Brucella was introduced into the Brucella-resistant RAW264.7 cell line to modulate the host's resistance to Brucella infection.

9. The application of the biological product that silences or knocks out the ID2 gene according to claim 8 in regulating host resistance to Brucella infection, characterized in that, The Brucella species is Brucella-M5.

10. The application of the biological product that silences or knocks out the ID2 gene according to claim 1 in regulating host resistance to Brucella infection, characterized in that, The amino acid sequence of the protein encoded by the ID2 gene has a protein accession number of P41136 in the UniProt database.

Images