Construction method of Tyrobp gene knockout macrophage cell strain and application of Tyrobp gene knockout macrophage cell strain in Brucella infection

Abstract

The invention belongs to the technical field of biology, and particularly relates to a construction method of a Tyrobp gene knockout macrophage cell strain and application of the Tyrobp gene knockout macrophage cell strain in Brucella LPS induced inflammatory response. According to the invention, a CRISPR / Cas9 gene editing technology is combined with a lentivirus expression system, a Tyrobp gene knockout RAW264.7 macrophage cell strain (KO Tyrobp-RAW264.7) is successfully constructed, and a gene knockout effect is verified through a genome sequencing method and a Western blot method. Tests show that the gene knockout cell strain shows remarkable weakening of the phagocytosis function while maintaining the normal multiplication capacity. After Brucella LPS treatment, a KO Tyrobp-RAW264.7 cell strain shows that the expression levels of inflammatory factors and NF [kappa] B are remarkably improved, which indicates that the Tyrobp gene has an important regulation effect in the inflammatory reaction induced by the Brucella LPS. The invention provides a new experimental model and a technical platform for deeply researching the antibacterial immune mechanism of Brucella, and has important application value in the field of biomedicine.

Description

Technical Field

[0001] The invention discloses a method for obtaining knockout mice Tyrobp The invention discloses a method for a gene cell strain and application of the cell strain in research on Brucella infection, belonging to the field of biotechnology. Background Art

[0002] Brucella ( Brucella ) is a facultative intracellular parasite that can preferentially infect the host's macrophages and regulate the host immune response through a series of complex mechanisms to maintain its survival and replication in the host. Brucella interacts with the lipid raft microdomains of macrophages through its lipopolysaccharide (LPS), promoting its internalization and forming phagosomes. The phagosomes then fuse with lysosomes to form phagolysosomes, in which most Brucella are killed. However, about 10% of Brucella can still survive 48 hours after infection and form an ecological niche in the endoplasmic reticulum of macrophages for replication and evasion of the bactericidal effects of host cells. Through this mechanism, Brucella can avoid being cleared and continue to infect other cells of the host.

[0003] Brucella can not only survive through physical mechanisms, but also help it survive for a long time by regulating the host immune response, especially the innate immune response. Specifically, after infection with Brucella, the production of multiple proinflammatory cytokines, such as TNF-α, IL-12, and IFN-γ, is inhibited, thereby weakening the immune activation ability of macrophages and reducing their ability to clear bacteria. For example, when Brucella infects human macrophages, it can inhibit the secretion of TNF-α and prevent macrophages from producing effective bactericidal activity. These regulatory effects can be carried out through the NF-κB pathway. NF-κB is an important transcription factor that regulates macrophage polarization. After Brucella infects macrophages, it can inhibit the activation of macrophages by upregulating the expression of negative regulatory proteins of NF-κB, thereby affecting the host's ability to clear bacteria.

[0004] In recent years, governments at all levels and relevant functional departments have attached great importance to the prevention and control of brucellosis, and have taken purification measures to reduce the incidence of the disease. However, with the continuous expansion of the scale of breeding and the gradual increase in the circulation of live animals, the incidence of brucellosis has shown an upward trend, seriously affecting and restricting the healthy development of animal husbandry. Brucella is an intracellular parasite that maintains its own survival by evading the host's immune mechanism after infection, and can survive for a long time in the mammalian host. The vaccine has poor protection effect, and there are residual virulence and interference with conventional serological tests. Improving the disease resistance of the population through disease-resistant breeding through precise molecular design is an effective means of preventing and controlling brucellosis, but due to insufficient understanding of the pathogenic mechanism of Brucella, breeding progress is slow.

[0005] The immune-related molecule TYROBP plays an important role in myeloid cells such as macrophages and natural killer cells. TYROBP mediates intracellular signal transduction through the immunoreceptor tyrosine-based activation motif (ITAM) and participates in the regulation of immune cell responses. Studies have shown that the loss of TYROBP can enhance the immune response of macrophages to a variety of bacteria, and the production of inflammatory factors TNF-α and IL-6 is significantly enhanced, indicating that TYROBP plays an important negative regulatory role in the activation of macrophages and bacterial clearance.

[0006] The interaction between these bacteria and the host immune system provides a basis for a deeper understanding of Brucella infection, and also provides a potential target for developing effective treatment strategies. The present invention, on the basis of the existing, establishes a novel immune regulation strategy for Brucella infection, which enhances the immune response of host macrophages by regulating TYROBP and related signaling pathways, thereby effectively removing Brucella infection. The present invention has the characteristics of strong innovation, high novelty, good safety, etc., and provides a reliable technical platform for subsequent antibacterial research. Summary of the invention

[0007] The object of the present invention is to provide a Tyrobp The construction method of gene knockout macrophage cell line and its application in the study of Brucella-induced inflammatory response, the specific technical scheme is as follows: The present invention provides a Tyrobp Method for constructing knockout macrophage cell lines using CRISPR / Cas9 technology Tyrobp The macrophage cell line containing the gene, the construction method comprises: searching the NCBI database for mouse Tyrobp Considering the short exon sequence, the first and third exon sequences of the genome were selected to design three sgRNAs, including the protospacer adjacent motif ( PAM ). The designed sgRNA sequences are as follows: SgRNA #1: ACGGAAGAACAGTCGCATCT TGG SgRNA#2: GACTGTTCTTCCGTGAGCCC TGG SgRNA#3: TGGGGGCTCTGGAGCCCTCC TGG Then design the DNA oligo primer of sgRNA sequence, anneal it and connect it with LentiCRISPR vector after restriction digestion to construct TyrobpLentiCRISPR-sgRNA, a lentiviral knockout vector for gene. After the recombinant vector is transformed into competent cells, a single colony is picked and expanded for culture to extract the plasmid. After the sequencing is correct, the virus supernatant is collected after transfection into the tool cells, and after centrifugation and filtration, it is infected with the culture medium in a certain ratio to the target cells to obtain Tyrobp Knockout macrophage cell lines.

[0008] Further, the DNA oligo primer sequence is as follows: SgRNA#1-F: caccgACGGAAGAACAGTCGCATCT SgRNA#1-R: aaacAGATGCGACTGTTCTTCCGTc SgRNA#2-F: caccgGACTGTTCTTCCGTGAGCCC SgRNA#2-R: aaacGGGCTCACGGAAGAACAGTCc SgRNA#3-F: caccgTGGGGGCTCTGGAGCCCTCC SgRNA#3-R: aaacGGAGGGCTCCAGAGCCCCCAc Furthermore, the annealing reaction system of the DNA oligo primer is as follows: SgRNA#1-F or SgRNA#2-F or SgRNA#3-F, 1 μL; SgRNA#1-R or SgRNA#2-RF or SgRNA#3-R, 1 μL; 10×T4 Buffer (Vazyme), 1 μL; ddH 2 O, 7 μL. Annealing program: 95°C, maintained for 3 minutes; cooled to 85°C at a rate of 2°C per second; maintained at 85°C for 1 minute; cooled to 75°C at a rate of 0.3°C per second; maintained at 75°C for 1 minute; cooled to 25°C at a rate and time from 85°C to 75°C; kept at 4°C. The annealing product was obtained.

[0009] Furthermore, the endonuclease used for the LentiCRISPR vector digestion is BnB Ⅰ (Neb Biolabs).

[0010] Furthermore, the ligation system used was: annealed product, 1.5 μL; LentiCRISPR vector after enzyme digestion, 100 ng; 10×T4 Buffer, 2 μL; T4 ligase, 1 μL; ddH 2 The volume was filled to 20 μL with 1% O. The ligation procedure was as follows: incubate at 22°C for 15 minutes.

[0011] Furthermore, the competent cells are DH5α.

[0012] Furthermore, the tool cells are HEK293T cells.

[0013] Furthermore, the plasmid transfection method is liposome transfection.

[0014] Furthermore, the Tyrobp The method for constructing gene knockout macrophages is as follows: Cell transfection and screening Establishment and verification of monoclonal cell lines Knockout (KO) Tyrobp Biological function detection of cell lines.

[0015] Further, the method in step a) is specifically as follows: the correctly sequenced plasmid is co-transfected with PAX2 and VSVG packaging plasmids into HEK293T cells, and the viral supernatant is collected 48 hours later. The viral liquid is filtered using a 0.45 μm filter membrane, mixed with the culture medium at a ratio of 1:1, and infects RAW264.7 cells. After 48 hours of infection, stably transfected hybrid cells are obtained by puromycin selection.

[0016] Furthermore, the method for establishing a monoclonal cell line in step b) is: the mixed cells obtained in step a) are cultured in a 96-well plate (Thermo), with a single cell in each well. The verification method is: genome sequencing and Western blot.

[0017] Furthermore, the biological function detection method in step c) is to detect the proliferation ability and phagocytosis ability of macrophages. The proliferation ability detection method is 5-ethynyl-2'-deoxyuridine (EDU) staining; the phagocytosis ability detection method is to use green fluorescent labeled Escherichia coli (pHrodo™ Green E. coli BioParticles™ conjugates) were used for detection.

[0018] The present invention also provides a method for establishing a Brucella LPS stimulation test scheme, wherein an optimal treatment concentration is determined through a Brucella LPS concentration gradient test, and the optimal concentration is used to treat macrophage RAW264.7 for a period of time, and the changes in the Brucella LPS-induced inflammatory response are detected.

[0019] Further, the Brucella LPS is Brucella bovis ( Brucella abortus S19) LPS.

[0020] Furthermore, the optimal concentration of Brucella LPS is 5 μg / mL.

[0021] Furthermore, the Brucella LPS treatment time is 4 hours.

[0022] Furthermore, the methods for detecting cell changes were transcriptome, relative fluorescence quantitative PCR and Western blot, and the detection indicators were the total protein and phosphorylated protein levels of inflammatory factors and SYK and NFκB.

[0023] Furthermore, the Tyrobp Gene knockout of macrophages can enhance the immune response of cells by inducing changes in the phosphorylation levels of SYK and NFκB proteins, promoting the continuous production of proinflammatory cytokines, thereby enhancing the ability to clear bacteria.

[0024] The advantage of the present invention is that a stable Tyrobp The gene knockout cell line provides a reliable experimental model for studying Brucella LPS-induced inflammatory response. At the same time, it clarifies the molecular mechanism of TYROBP in Brucella LPS-induced inflammatory response, providing a new target for the molecular mechanism research and treatment of brucellosis, which has important theoretical and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 Map of the constructed LentiCRISPR-sgRNA lentiviral vector; Figure 2 Schematic diagram of the knockout site genome and sequencing results; Figure 3 Western blot detection results of TYROBP in WT and KO group cells; Figure 4 Cell morphology of WT group and KO group; Figure 5 The results of EdU staining of cells in the WT and KO groups; Figure 6 The phagocytosis test results of cells in the WT group and KO group; Figure 7 The changes of inflammatory factors in two cell lines treated with Brucella LPS at different time gradients; Figure 8 The transcriptome sequencing results show the enrichment pathway of down-regulated differentially expressed genes in the KO group compared with the WT group after the two cells were treated with Brucella LPS for 4 h. Fig. 9These are the Western blot results of proteins in the WT and KO groups after cells were treated with Brucella LPS for 4 h.

[0027] The above drawings illustrate Tyrobp The effects of gene knockout on cell function and immune response provide important experimental basis for further research. DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Example 1

[0029] Embodiment 1 of the present invention provides a Tyrobp Method for constructing gene knockout macrophage cell lines.

[0030] 1.Tyrobp Design of sgRNA: Selection of mice Tyrobp The sgRNA was designed for the first and third exons of the gene (Gene ID: 22177), and the sequence including the PAM sequence is as follows: SgRNA#1: ACGGAAGAACAGTCGCATCTTGG (SEQ ID No.1) SgRNA#2: GACTGTTCTTCCGTGAGCCCTGG (SEQ ID No.2) SgRNA#3: TGGGGGCTCTGGAGCCCTCCTGG (SEQ ID No. 3).

[0031] 2. Construction of LentiCRISPR-sgRNA lentiviral vector.

[0032] 2.1 According to the designed sgRNA, design DNA oligos for synthesizing sgRNA. The primer sequences are as follows: SgRNA#1-F: caccgACGGAAGAACAGTCGCATCT (SEQ ID No.4) SgRNA#1-R: aaacAGATGCGACTGTTCTTCCGTc (SEQ ID No.5) SgRNA#2-F: caccgGACTGTTCTTCCGTGAGCCC (SEQ ID No.6) SgRNA#2-R: aaacGGGCTCACGGAAGAACAGTCc (SEQ ID No.7) SgRNA#3-F: caccgTGGGGGCTCTGGAGCCCTCC (SEQ ID No.8) SgRNA#3-R: aaacGGAGGGCTCCAGAGCCCCCAc (SEQ ID No.9) The above-mentioned primers were synthesized using ddH 2 O to a concentration of 10 μM.

[0033] Annealing reaction system: SgRNA#1-F or SgRNA#2-F or SgRNA#3-F, 1 μL; SgRNA#1-R or SgRNA#2-RF or SgRNA#3-R, 1 μL; 10×T4 Buffer (Vazyme), 1 μL; ddH 2 O, 7 μL.

[0034] The annealing procedure is: 95°C, maintained for 3 minutes; cooled to 85°C at a rate of 2°C per second; maintained at 85°C for 1 minute; cooled to 75°C at a rate of 0.3°C per second; maintained at 75°C for 1 minute; cooled to 25°C at a rate and time from 85°C to 75°C; kept at 4°C to obtain the annealing product.

[0035] 2.2 Use of restriction enzymes BnB Ⅰ (Neb Biolabs) Enzyme digestion of LentiCRISPR vector, the enzyme digestion system is: LentiCRISPR vector, 1 μg; BnB Ⅰ, 1μL; 10×Buffer, 5μL; ddH 2 O, make up to 50 μL. The enzyme digestion program is: 55°C, 1 hour. Use 1% agarose gel electrophoresis, and then use DNA purification and recovery kit (Tiangen) to recover.

[0036] 2.3 The recovered digested vector and annealed product were connected. The connection system was: annealed product, 1.5 μL; LentiCRISPR vector after digestion, 100 ng; 10×T4 Buffer, 2 μL; T4 ligase, 1 μL; ddH 2 The volume was filled to 20 μL with 1% O. The ligation procedure was: 22°C, 15 minutes.

[0037] 2.4 The ligation product was transformed into DH5α cells. The transformation system was as follows: the volume of the ligation product was one-tenth of the volume of the competent cells. Transformation procedure: ice bath for 30 minutes, 42°C, 90 seconds; ice bath for 5 minutes. The transformation product was supplemented with 800 μL of liquid culture medium without antibiotics. After culturing at 37°C constant temperature shaker at 180 rpm for 1 hour, the bacteria were collected by centrifugation and spread on a plate containing ampicillin. Incubate at 37°C constant temperature incubator for 14 hours. Pick a bacterial monoclone and culture it in liquid culture medium containing ampicillin at 37°C constant temperature shaker at 180 rpm for 16 hours.

[0038] 2.5 Use the Endotoxin-free Plasmid Extraction Kit (Tian Gen) to extract the endotoxin-free plasmid and send it to sequencing. The vector with the correct sequencing should be stored at -20℃ (the plasmid map is shown in Figure 1 as shown).

[0039] Lentiviral packaging.

[0040] 3.1 Cell preparation: 293T cells within the fifth generation were inoculated in a 60 mm culture dish in DMEM (Corning) complete medium (supplemented with 10% fetal bovine serum) at 37°C and 5% CO. 2 When the cell growth density reaches about 80%, discard the culture supernatant and replace with 3 mL DMEM basal medium, and starve the cells at 37°C for 1 hour.

[0041] 3.2 Plasmid transfection: Calculate and prepare a 9 μg plasmid mixture according to the ratio of lentiviral plasmid containing SgRNA sequence: PAX2: VSVG = 4: 3: 2. Gently mix 27 μL PEI (Biyuntian) with 200 μL Opti MEM™ and let it stand for 5 minutes. Then, mix 9 μg plasmid mixture with 200 μL Opti MEM™, add it to the PEI-OptiMEM™ mixture, mix it gently and let it stand for 20 minutes.

[0042] Replace the DMEM basal medium in the culture dish with 4 mL of DMEM complete medium, and slowly and evenly add the plasmid-PEI-OptiMEM™ mixture onto the cell surface, minimizing the shaking of the liquid. Place the cells in a 5% CO 2 , and continue culturing in a 37°C constant temperature incubator.

[0043] 3.3 Culture and collection: 12 hours after plasmid transfection, replace with DMEM complete medium and continue culture. 48 hours after medium replacement, collect the supernatant, centrifuge at 2000 rpm for 5 minutes, and filter through a 0.45 μm filter to obtain the lentiviral solution containing the SgRNA sequence.

[0044] Monoclonal cell screening.

[0045] 4.1 Cell preparation: RAW264.7 cells within the third generation were seeded in 35 mm culture dishes (Thermo) and cultured in 5% CO 2 , culture in a 37°C constant temperature incubator, and infect when the cell growth density reaches about 30%.

[0046] 4.2 Virus infection: The filtered virus solution was mixed with DMEM complete medium at a ratio of 1:1, and 2 μL Polybrene (10 mg / mL, Sigma Aldrich) was added to infect RAW264.7 cells. The cells were placed in 5% CO 2 , and infect in a 37°C constant temperature incubator for 12 hours.

[0047] 4.3 Puromycin selection: After infection, the cells were cultured continuously until the cell density reached 80%. 8 μg / mL Puromycin was used to select the cells for 12 hours, and then the medium was replaced with DMEM complete medium. After the cells recovered, the selection was repeated.

[0048] 4.4 Monoclonal cell line screening: The screened cells were resuspended in DMEM complete medium and diluted to an appropriate concentration. A certain amount of the cell suspension was inoculated in a 96-well plate (Thermo) and the presence of single cells was confirmed under a microscope. After supplementing with 100 μL of DMEM complete medium, the culture was continued. After the single cells were amplified to a sufficient number, the cell pellet was collected for genome sequencing.

[0049] Genomic detection of knockout sites.

[0050] 5.1 Genomic extraction: Monoclonal cells were expanded and cultured in 12-well plates (Thermo). When the cell density reached 80%, the cells were collected by centrifugation at 1000 rpm for 3 minutes. Genomic DNA was extracted according to the instructions of the Blood / Cell / Tissue Genomic DNA Extraction Kit (Tiangen).

[0051] 5.2 PCR amplification: PCR amplification was performed using amplification primers and PrimeSTAR® Max DNA Polymerase (Takara). The amplified product was subjected to 2% agarose gel electrophoresis, and the target band was cut out and recovered using a DNA purification recovery kit and sequenced. The sequencing primers were the upstream primers of each.

[0052] 5.3 Analysis of sequencing results: cells with altered sequences between the 3rd and 4th bases in the 5' direction of the PAM sequence were selected as positive cells. Figure 2 As shown, the knockout group cells TyrobpSeven base pairs were inserted into the third exon of the genome, and the KO group cells Tyrobp The nucleotide sequence of the core coding region of the gene was changed to: atgggggctctggagccctcctggtgccttctgttccttcctgtcctcctgactgtgggaggattaagtcccgtacaggcccagagtgacactttcccaagatgcgactgttcttccgtgagtgtacttccctggtgtactggctgggattgttctgggtga (SEQ ID No.10); the amino acid sequence of the TYROBP protein was changed to: MGALEPSWCLLFLPVLLTVGGLSPVQAQSDTFPRCDCSSVSVLPWCTGWDCSG* (wherein * is a stop codon) (SEQ ID No.11).

[0053] Knockout was verified by Western blot.

[0054] 6.1 Cell lysis: Culture the cells in a 60 mm culture dish. When the cell density reaches more than 80%, discard the culture medium, rinse the cells twice with pre-cooled PBS, and collect the cells by centrifugation. Use RIPA lysis buffer (Biyuntian) to lyse the cells, add a mixture of protease inhibitors (Solebo) and a mixture of phosphatase inhibitors (Solebo), sonicate at 300 W for 15 seconds, let stand for 15 seconds, and cycle 3 times. Centrifuge at 4°C and 12,000 rpm for 10 minutes, and collect the supernatant.

[0055] 6.2 Protein sample preparation: Add 5× protein loading buffer (Epizyme) to the supernatant, boil at 100°C for 10 minutes, and store the sample at -80°C.

[0056] 6.3 SDS-PAGE and transfer: SDS-PAGE was performed using the Yisheng Bio 10% PAGE gel rapid preparation kit. AR rapid transfer solution (AccuRef Scientific) was used, and the transfer was carried out at a constant current of 400 mA for 30 minutes. The PVDF membrane was from Sigma.

[0057] 6.4 Antibody incubation and development: Block with 8% skim milk for 2 hours, incubate with primary antibody at 4°C overnight, incubate with secondary antibody at room temperature for 1 hour, develop and expose. The antibodies used and the dilution ratios are as follows: Anti-TYROBP (Abcam), 1:1500; Anti-GAPDH (Scicrest biotech), 1:5000; HRP Conjugated AffiniPure Goat Anti-rabbitIgG (H+L)(Boster Biological Technology), 1:5000; HRP Conjugated AffiniPure GoatAnti-mouse IgG (H+L) (Boster Biological Technology), 1:5000. The TYROBP protein in the knockout group cells is almost completely unexpressed. The results are as follows Figure 3 shown. Example 2

[0058] Tyrobp Changes in physiological functions of gene knockout macrophage lines.

[0059] 1. EdU staining detection Tyrobp Effects of gene knockout on RAW264.7.

[0060] 1.1 Cell fixation and processing for sequencing of correct knockouts Tyrobp Monoclonal cells of genes (such as Figure 4 Expanded culture.

[0061] When the cells were in the logarithmic growth phase, they were washed three times with PBS and fixed with 4% PFA at room temperature for 15 minutes. 5 mg / mL glycine solution was used to remove aldehydes at room temperature for 15 minutes, and 0.5% Triton X-100 solution was used to perforate the cell membrane at room temperature for 15 minutes.

[0062] 1.2 Staining and observation: Cell-Light EdU Apollo567 In Vitro Kit (Raybo Biotech) was used for EDU staining. The staining process is detailed in the instructions. After staining, randomly select the field of view under a fluorescence microscope to take pictures and record the results. The results show that knockout Tyrobp The gene does not affect the proliferation ability of RAW264.7 (such as Figure 5 as shown).

[0063] 2. Tyrobp Effects of gene knockout on the phagocytic function of RAW264.7.

[0064] 2.1 Cell preparation: The cells were inoculated into 24-well plates (Thermo) and cultured. When the cell density reached about 70%, the medium was replaced with DMEM basal medium.

[0065] 2.2 Phagocytosis assay: Cells were incubated with 1 mg / mL pHrodo™ Green E. coli BioParticles™ conjugates (Thermo Fisher Scientific) were incubated at 37°C in the dark for 3 hours. The ratio of cells emitting green fluorescence between wild-type cells and knockout cells was observed under a fluorescence microscope, photographed, and calculated. The results showed that Tyrobp The deletion of the gene will cause the phagocytic ability of RAW264.7 cells to be weakened (such as Figure 6 ), which may indicate that the deletion of this gene can reduce the phagocytosis of Brucella by macrophages to a certain extent and reduce the intracellular bacterial load. Example 3

[0066] Detection Tyrobp Response of knockout macrophage cell lines to Brucella LPS stimulation.

[0067] In this example, Brucella LPS was diluted with DMEM basal medium and RAW264.7 (wild-type and knockout cells) was treated at a concentration of 5 μg / mL. When the cells grew to 80%, the cells were collected for RNA and protein extraction to detect changes in the expression levels of inflammatory factors and the like in the two groups of cells.

[0068] 1. RNA extraction and reverse transcription, and fluorescence quantitative PCR were used to detect changes in inflammatory factors.

[0069] 1.1 RNA extraction: Wild-type and knockout RAW264.7 cells were inoculated in 35 mm culture dishes. When the cell density reached 80%, 5 μg / mL Brucella LPS (Baojilan Biotech) was replaced and incubated at 37°C for 4 hours. The culture medium was discarded, washed twice with pre-cooled PBS, lysed with 1 mL RNAiso (Takara) for 5 minutes, and the lysate was collected.

[0070] 1.2 RNA purification: Add 200 μL of pre-cooled chloroform to each tube of lysate, vortex for 15 seconds, and place on ice for 15 minutes. Centrifuge at 4°C and 12,000 rpm for 15 minutes, take the colorless upper layer and mix it with an equal volume of pre-cooled isopropanol, and place on ice for 30 minutes. Centrifuge at 4°C and 12,000 rpm for 15 minutes, discard the supernatant, wash the precipitate with 75% ethanol, repeat once, centrifuge again at the same speed for 3 minutes, aspirate the residual liquid, ventilate and dry for 5 minutes, and resuspend the RNA precipitate with 20-30 μL DEPC water.

[0071] 1.3 RNA detection: The concentration and purity of RNA were detected using a micro-UV spectrophotometer (Thermo). The A260 / A280 ratio was qualified when it was between 1.8 and 2.1.

[0072] 1.4 Reverse transcription and relative fluorescence quantitative PCR: 2 μg RNA was reverse transcribed using HiScript III RT SuperMix for qPCR (+gDNA wiper) (Novozyme) and diluted to 20 ng / μL for fluorescence quantitative PCR. Fluorescence quantitative PCR was performed using the ChamQSYBR qPCR Master Mix Kit (Novozyme) according to the instructions. The results were analyzed using Bio-Rad CFX Manager 3.1 software, and the data were presented in GraphPad Prism v9.0. The two groups of data were compared using the Student's t test. P The difference was significant when the value was <0.05. The RNA levels of inflammatory factors TNFα and IL6 in wild-type cells and knockout cells were treated with 5 μg / mL Brucella LPS for different time periods (0h, 4h, 8h, and 12h). Figure 7 As shown, knockout Tyrobp After gene knockout, RAW264.7, although the expression of inflammatory factors in the knockout group cells was lower than that in the control group at 4 hours, its inflammatory factors were maintained at a high level as a whole over the entire timeline.

[0073] 1.5 Primer sequences are: m- Gapdh -F: GGTGTCCGTTGTGGATCTGA (SEQ No.12) m- Gapdh -R: TGAAGTCGCAGGAGACAACC (SEQ No.13) m- Tnf -F: GACCCTCACACTCAGATCATCTTC (SEQ No.14) m- Tnf -R: CACGTAGTCGGGGCAGCCTTG (SEQ No.15) m- Il6 -F: GTATGAACAACGATGATGCAC (SEQ No.16) m- Il6 -R: CTCCAGAAGACCAGAGGAAA (SEQ No. 17).

[0074] 2. RNA sequencing.

[0075] 2.1 Sample preparation: Wild-type and knockout RAW264.7 cells were inoculated in 60 mm culture dishes. When the cells grew to 80%, 1 mL of RNAiso lysis reagent was used to lyse the cells. The lysate was pipetted until it was clear, and then quickly frozen in liquid nitrogen and stored at -80°C.

[0076] 3.2 RNA sequencing: RNA sequencing services are provided by BGI, and some of the results are as follows Figure 8 As shown in Figure 2, similar to the results of relative fluorescence quantitative PCR, the differentially regulated genes downregulated in the knockout group were mainly enriched in immune-related pathways. In addition, they were also enriched in pathways such as proteolysis, indicating that TYROBP may also play a certain role in proteolysis (such as Figure 8 as shown).

[0077] 3.Western blot was used to detect the expression of total protein and phosphorylated protein of SYK and NF-κB.

[0078] The steps were the same as step 6 in Example 2. The antibodies used and the dilution ratios were as follows: Anti-TYROBP (Abcam), 1:1500; Anti-GAPDH (Scicrest biotech), 1:5000; Anti-NF-κB (CST), 1:1000; Anti-pNF-κB (CST), 1:1000; Anti-SYK (Proteintech), 1:1000; Anti-pSYK (Immunoway), 1:1000; HRP Conjugated AffiniPure Goat Anti-rabbit IgG (H+L) (Boster Biological Technology), 1:5000; HRP Conjugated AffiniPure Goat Anti-mouse IgG (H+L) (Boster Biological Technology), 1:5000.

[0079] The results are as follows Fig. 9 As shown, the total level of SYK protein increased, but its phosphorylation level weakened, indicating the existence of compensatory balance regulation; the phosphorylation level of NFκB was significantly enhanced.

[0080] These results suggest that TYROBP deficiency leads to dysregulation of the SYK-NFκB axis, driving the sustained production of proinflammatory cytokines and thus enhancing bacterial clearance.

[0081] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Obtaining knockout mice Tyrobp A method for constructing a RAW264.7 cell line expressing a gene, characterized in that: The following steps are involved: a) Using CRISPR / Cas9 gene editing technology and lentiviral vector construction Tyrobp Knockout RAW264.7 cell line; b) Verifying through genome sequencing that the cell line has 7 base pairs (bp) inserted in the third exon region, resulting in the premature appearance of the stop codon and shortening the amino acid sequence from 115 to 54; c) confirming that TYROBP protein is almost completely depleted by protein level detection; d) The cell line showed increased activation of the NFκB signaling pathway during Brucella LPS infection.

2. A verification Tyrobp The method for enhancing the anti-infection function of genes is characterized in that, The following steps are involved: a) Build Tyrobp Knockout RAW264.7 cell line; b) treating the cell strain with an effective amount of Brucella LPS; c) detecting the level of inflammatory factors in the cell line.

3. The method according to claim 1, characterized in that The small guide RNA (sgRNA) sequence used in the CRISPR / Cas9 gene editing technology is selected from any of the following (including the protospacer adjacent motif at the end): SgRNA#1: 5'-ACGGAAGAACAGTCGCATCTTGG-3' SgRNA#2: 5'-GACTGTTCTTCCGTGAGCCCTGG-3' SgRNA#3: 5'-TGGGGGCTCTGGAGCCCTCCTGG-3'.

4. The method according to claim 1, characterized in that Said Tyrobp Gene knockout of RAW264.7 cell line was achieved by lentiviral vector transfection.

5. The method according to claim 2, characterized in that The stimulation concentration of the Brucella LPS is 1 μg / mL to 5 μg / mL.

6. The method according to claim 2, characterized in that The stimulation time of the Brucella LPS is 4 hours or within the range of ±4 hours.

7. The method according to claim 2, characterized in that The inflammatory factors include TNF-α and IL-6.

8. The method according to claim 1 Tyrobp The use of gene knockout RAW264.7 cell line in screening anti-infective drugs or studying the regulatory mechanism of NFκB signaling pathway.

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