Application of tmem214 gene as a target in preparation of medicine for treating brucella infection

Abstract

The application provides a method for treating brucella infection by using a gene as a target point TMEM214 The application belongs to the technical field of genetic engineering and relates to application of a gene as a target point in preparation of a medicine for treating brucella infection TMEM214 The gene sequence is shown as SEQ ID NO: 1. Experiments prove that, after RAW264.7 macrophages are infected by bovine brucella A19, expression of the TMEM214 protein is significantly up-regulated; and knockout of the gene can increase the survival number of intracellular brucella. TMEM214 Animal experiments show that, compared with wild-type mice, the spleen brucella load of TMEM214 homozygous knockout mice is significantly increased after infection, proving that the gene can inhibit intracellular colonization and proliferation of brucella. TMEM214 The application provides a new target point for genetic breeding of livestock against brucellosis, development of a target drug and a new vaccine against brucella, opens up a new idea for clinical prevention and treatment of brucellosis, and is favorable for construction of a comprehensive prevention and control system.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the application of the TMEM214 gene as a target in the preparation of drugs for treating Brucella infection. Background Technology

[0002] Brucellosis, commonly known as brucellosis, is a globally prevalent zoonotic bacterial infectious disease caused by Brucella. In humans, infection often manifests as a prolonged, recurrent, and chronic illness, with severe cases leading to loss of working capacity. In livestock, infection easily causes numerous abortions, infertility, sterility, and stillbirths, resulting in a significant decline in livestock numbers. Currently, brucellosis outbreaks have been reported in over 170 countries and regions worldwide, with approximately 500,000 new cases annually, posing a significant impact on global economic development and public health security. In my country, over the past two decades, with the expansion of livestock and poultry farming and the increasingly frequent circulation of livestock products, human brucellosis infections have shown a significant upward trend. According to statistics from the Chinese Center for Disease Control and Prevention, 62,099 new human brucellosis cases were reported nationwide in 2025, indicating a still serious epidemic situation.

[0003] Brucella, an intracellular parasite, is the root cause of the difficulty in treating brucellosis. This bacterium can invade and colonize immune cells such as macrophages, replicating intracellularly and effectively evading antibiotics and the host's immune system. Conventional antibiotics struggle to penetrate cell membranes to kill the pathogen; therefore, clinical treatment requires specific antibiotics with good cell penetration capabilities, employing a combination therapy strategy with adequate dosage and long treatment duration. Consequently, the clinical treatment of brucellosis faces numerous challenges, necessitating more effective diagnostic and treatment strategies and control measures.

[0004] TMEM214 is a key protein located on the endoplasmic reticulum membrane. Its core function is to act as an "anchor" to recruit and activate caspase-4 during endoplasmic reticulum stress, thereby initiating apoptosis. Therefore, TMEM214 is a "death switch" protein that plays a crucial role in the endoplasmic reticulum stress pathway. However, whether the TMEM214 gene / protein affects Brucella infection has not yet been reported in studies. Summary of the Invention

[0005] In view of this, the present invention provides the application of the TMEM214 gene as a target in the preparation of drugs for treating Brucella infection, in order to solve the above problems.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides the application of the TMEM214 gene and / or the protein encoded by the TMEM214 gene as a target in the preparation of drugs for the prevention and / or treatment of brucellosis; the nucleotide sequence of the TMEM214 gene is shown in SEQ ID NO:1, and the amino acid sequence of the protein encoded by the TMEM214 gene is shown in SEQ ID NO:2.

[0008] Preferably, the expression of the TMEM214 protein or TMEM214 gene is promoted.

[0009] Preferably, Brucella abortus strain A19 is used.

[0010] The present invention also provides a medicament for treating brucellosis, comprising an active ingredient capable of upregulating TMEM214 gene expression or enhancing TMEM214 protein activity, and a pharmaceutically acceptable carrier, excipient or adjuvant.

[0011] Preferably, the active ingredient of the drug is any one or more of the following: TMEM214 gene overexpression vector, TMEM214 recombinant protein, and TMEM214 protein agonist.

[0012] The present invention also provides a TMEM214 gene knockout RAW264.7 cell line, which is constructed by targeting and knocking out the 5' end sequence of the TMEM214 gene open reading frame using the CRISPR-Cas9 system. The sgRNA targeting sequence is shown in SEQ ID NO:3 and SEQ ID NO:6.

[0013] This invention provides a TMEM214 homozygous knockout mouse model, in which exons 3 to 17 of the mouse TMEM214 gene are knocked out using Cas9 / gRNA technology, resulting in the loss or functional inactivation of TMEM214 protein; compared with wild-type mice, homozygous knockout mice show a significant increase in splenic bacterial load after infection with Brucella.

[0014] Preferably, the Cas9 / gRNA binding target site sequence is shown in SEQ ID NO:9 and SEQ ID NO:10.

[0015] This invention also provides the application of the TMEM214 gene knockout RAW264.7 cell line or the TMEM214 homozygous knockout mouse model in studying the intracellular infection mechanism of Brucella and screening anti-brucellosis drugs.

[0016] By adopting the above technical solution, the present invention has the following beneficial effects: The TMEM214 gene sequence of the present invention is shown in SEQ ID NO:1. Experiments have confirmed that after infection of RAW264.7 macrophages with Bovine Brucella A19, the expression of TMEM214 protein is significantly upregulated; knocking out the TMEM214 gene increases the number of surviving Brucella bacteria in the cells. Animal experiments show that compared with wild-type mice, the amount of Brucella bacteria in the spleen of TMEM214 homozygous knockout mice is significantly increased after infection, proving that TMEM214 can inhibit the intracellular colonization and proliferation of Brucella. The present invention provides a novel target for genetic breeding of livestock against brucellosis, the development of anti-brucellosis targeted drugs and novel vaccines, opens up new ideas for the clinical prevention and treatment of brucellosis, and is conducive to the construction of a comprehensive disease prevention and control system. Attached Figure Description

[0017] Figure 1 The values ​​represent the TMEM214 protein levels before and after Brucella infection; Mock is the blank control, and 4h, 24h, and 48h represent the duration of Brucella infection; the 77kDa band represents the TMEM214 protein, and the 42kDa band represents β-actin, which is the internal reference protein.

[0018] Figure 2 Protein levels in TMEM214 knockout cell lines were identified; WT: wild-type RAW264.7 cells; KO: TMEM214 knockout cells; β-actin was used as an internal control for sample loading.

[0019] Figure 3 Effect of TMEM214 gene on intracellular survival of Brucella abortifacient A19 in RAW264.7 cells; WT: wild-type RAW264.7 cells; TMEM214 KO: TMEM214 knockout cells; ordinate: log CFU / mL, abscissa: infection time, *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.

[0020] Figure 4 Electrophoresis images for genotyping of TMEM214 homozygous knockout mice; the amplified fragment of wild-type mice is 318 bp, and the amplified fragment of TMEM214 homozygous knockout mice is 468 bp.

[0021] Figure 5 The effect of TMEM214 on the bacterial load of Brucella infection in the spleen of mice; WT mice were wild-type mice, and TMEM214 KO mice were TMEM214 knockout mice; 1×10⁻⁶ TMEM214 was administered via intraperitoneal injection. 6 CFU-induced abortion of Brucella A19, spleen viable bacterial load was measured 2 weeks after infection; *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001. Detailed Implementation

[0022] The nucleotide sequence of the TMEM214 gene is shown in SEQ ID NO:1:

[0023]

[0024] The amino acid sequence of the protein encoded by the TMEM214 gene is shown in SEQ ID NO:2:

[0025] (SEQ ID NO:2).

[0026] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0027] The experimental materials used in the embodiments of this invention are shown in Table 1.

[0028] Table 1 Test Materials

[0029] RAW264.7-Cas9 Guangzhou Yuanjing Biotechnology Co., Ltd. YC-C020-Cas9-H 293T Guangzhou Yuanjing Biotechnology Co., Ltd. YC-A006 Premium South American fetal bovine serum Suzhou Shuangru Biotechnology Co., Ltd. S711-001S Penicillin-Streptomycin Gibco 15140122 DMEM medium Gibco C11995500BT Puromycin Dihydrochloride Shanghai Beyotime Biotechnology Co., Ltd. ST551-10mg jetPRIME transfection reagent Polyplus Transfection 101000046 psPAX2 Addgene 12260 pMD2.G Addgene 12259 lentiGuide-sg-puro Addgene 52963 FastPure Cell / Tissue Total RNA Isolation Kit V2 Nanjing Novozymes Biotechnology Co., Ltd. RC112-01 RIPA lysis buffer (strong) Shanghai Beyotime Biotechnology Co., Ltd. P0013B Protease inhibitor mixture (universal, 100X) Shanghai Beyotime Biotechnology Co., Ltd. P1005 Prime STAR Max DNA Takara R045A HiScript III RT SuperMix for qPCR (+gDNA wiper) Nanjing Novozymes Biotechnology Co., Ltd. R323-01 Biotides 10% SDS-PAGE One-Step Gel Preparation Kit for PAGE Gel Preparation biotides WB2101 6× Protein Loading Buffer Beijing TransGen Biotech Co., Ltd. DL101-02 ProteinFind® Anti-DYKDDDDK Mouse Monoclonal Antibody Beijing TransGen Biotech Co., Ltd. HT201-01 ProteinFind® Goat Anti-Mouse IgG (H+L), HRP Conjugate Beijing TransGen Biotech Co., Ltd. HS201-01 Beta Actin Polyclonal antibody Proteintech 20536-1-AP CutSmart Buffer NEB B7204S BsmBI NEB R0580S T4 DNA Ligase Beijing TransGen Biotech Co., Ltd. FL101-01 5×T4 DNA Ligase Buffer Beijing TransGen Biotech Co., Ltd. GO101-01 DH5α competent cells Sangon Biotech (Shanghai) Co., Ltd. B528413-0100 <![CDATA[ddH2O]]> Beijing Solarbio Technology Co., Ltd. R1600 TSB Haibo Biotechnology Co., Ltd. HB4114-19 TSA Haibo Biotechnology Co., Ltd. HB0177-1

[0030] Brucella abortus strain A19 is described in the literature Wang, Shuyi et al. “The Genome Sequence of Brucella abortus vaccine strain A19 provides insights on its virulence attenuation compared to Brucella abortus strain 9-941.” Gene vol.830 (2022): 146521. doi:10.1016 / j.gene.2022.146521.

[0031] Example 1. Expression of TMEM214 gene / protein during Brucella infection.

[0032] In this embodiment, RAW264.7 macrophages were infected with Brucella bovis A19 at 4, 24, and 48 h. Uninfected RAW264.7 macrophages served as the control group. The TMEM214 protein level was detected by Western blotting in both the control group and the Brucella-infected experimental group. The specific steps are as follows:

[0033] (1) Wild-type RAW264.7 cells were divided into groups of 1×10⁻⁶ cells. 6 The cells were seeded at a density of 1 cell / mL in 6-well plates and cultured overnight in DMEM medium containing 2% FBS to allow the cells to adhere completely and reach approximately 80% confluence.

[0034] (2) Using Brucella abortus strain A19, add it to the corresponding adherent cells at a ratio of MOI=100. Centrifuge the infected plate at 400×g for 5 min and incubate it in a 37℃, 5% CO2 incubator for 1 h.

[0035] (3) Remove the bacterial culture medium and wash the cells three times with PBS to completely remove free bacteria that have not entered the cells. Then replace the medium with 2% FBS DMEM containing 50 μg / mL gentamicin and culture for 1 h to kill any remaining Brucella A19 outside the cells.

[0036] (4) Replace the culture medium with 2% FBS DMEM medium containing 25 μg / mL gentamicin and continue to culture. Terminate the culture when the infection has been present for 0h, 4h, 24h and 48h respectively. Remove the supernatant and wash the cells twice with pre-cooled PBS.

[0037] (5) Add 150 μL of RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors to each well and lyse on ice for 30 min. During lysis, repeatedly pipette the cells every 10 min to ensure complete lysis. Transfer the lysate to a 1.5 mL centrifuge tube and centrifuge at 12,000 rpm for 15 min at 4 °C. Carefully collect the supernatant containing total protein into a new centrifuge tube and place it on ice for later use. Add 5× protein loading buffer and heat in a 100 °C metal bath or boiling water for denaturation for 10 min. Then store at -80 °C for later use.

[0038] (6) Prepare a 10% SDS-PAGE gel, add 20 μg of total protein per well, run the stacking gel at 80V, and after the sample enters the separating gel, adjust the voltage to 120V and continue electrophoresis for about 90 minutes to separate the proteins by electrophoresis.

[0039] (7) Using the wet transfer method, electrophoresis was performed at a constant current of 200 mA for 90 min, and the protein imprints separated on the gel were transferred to a PVDF membrane with a pore size of 0.45 μm.

[0040] (8) Immerse the PVDF membrane after transfer in 5% skim milk powder (or 5% BSA) prepared with TBST and seal it on a shaker at room temperature for 2 hours to reduce nonspecific binding.

[0041] (9) Remove the blocking solution, add the TMEM214 specific primary antibody and β-actin internal control primary antibody diluted according to the ratio, and incubate overnight in a shaker at 4°C; wash the membrane 3 times with TBST the next day, 10 min each time.

[0042] (10) Add the corresponding horseradish peroxidase (HRP) labeled secondary antibody to the blocking dilution buffer, incubate at room temperature for 1 h, and then wash the membrane three times with TBST for 10 min each time to completely wash away the free secondary antibody.

[0043] (11) ECL ultrasensitive luminescent liquid was uniformly dropped onto the PVDF film, and the film was exposed and developed in a darkroom under a chemiluminescence imaging instrument. The gray value of the target band was quantitatively analyzed using ImageJ software.

[0044] (12) The results of the Western Blot are as follows Figure 1 As shown, the results indicate that, at the protein level, the expression of TMEM214 protein in RAW264.7 cells was significantly upregulated after infection.

[0045] Example 2. Construction of TMEM214 gene / protein monoclonal knockout cell line

[0046] (1) Wild-type RAW264.7-Cas9 cells in good condition were seeded in 6-well plates with 5 cell seeding density gradients. When the cell confluence reached 60% to 70% and the cells were in good condition after 24 to 48 hours of culture, complete medium containing different concentration gradients of puromycin (0.125 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL) was added for screening. The drug-containing medium was replaced every 24 to 48 hours and the screening was continued for at least 7 days.

[0047] (2) After screening, cells were stained with 0.04% trypan blue solution and their survival was counted under a microscope. Seven days after administration, all RAW264.7 cells died at a puromycin concentration of 0.5 μg / mL, thus determining that the working concentration for stable transfection screening was 0.5 μg / mL.

[0048] (3) Based on the sequence information of the TMEM214 gene, two sgRNA sites were designed near the 5' end of the open reading frame (ORF), and the corresponding cloning primers were synthesized. The sgRNA and sgRNA annealing primer sequences are shown below:

[0049] sgRNA1: 5'-atgcatcccaacctcaccaa-3' (SEQ ID NO: 3);

[0050] sgRNA1-F: 5'-atgcatcccaacctcaccaa-3' (SEQ ID NO: 4);

[0051] sgRNA1-R: 5'-ttggtgaggttgggatgcat-3' (SEQ ID NO: 5)

[0052] sgRNA2: 5'-atataggcaaagtccagaag-3' (SEQ ID NO: 6);

[0053] sgRNA2-F: 5'-atataggcaaagtccagaag-3' (SEQ ID NO: 7);

[0054] sgRNA2-R: 5'-cttctggactttgcctatat-3' (SEQ ID NO: 8);

[0055] Prepare the annealing system for the corresponding upstream and downstream primers: Mix 5 μL each of the leading and lagging strands (total 10 μL), and set the annealing program as follows: denature at 95 °C for 5 min; reduce to 85 °C at 2 °C / cycle (5 cycles, 0.7 s / cycle); then slowly reduce to 25 °C at 0.1 °C / cycle (600 cycles, 0.7 s / cycle); finally, incubate at 4 °C to obtain double-stranded sgRNA.

[0056] (4) Using BsmB I restriction endonuclease and T4 DNA ligase, the annealed sgRNA double-stranded fragment was cloned into the lenti-sg-puro lentiviral vector. The vector digestion system was as follows: plasmid linearization was performed using a 100 μL digestion reaction system containing 6 μg plasmid vector lenti-sg-puro, 10 μL 10× CutSmart Buffer and 3 μL BsmB I restriction endonuclease, and incubated overnight in a 55 ℃ water bath. After the digestion product was identified as completely digested by 1% agarose gel electrophoresis (120 V, 30 min), the gel was excised and the linearized vector was purified using a gel extraction kit.

[0057] Subsequently, recombination ligation was performed using a 10 μL T4 DNA ligation system. 2 μL of 5× T4 Ligase Buffer, 50 ng of linearized lenti-sg-puro vector (carrying the U6 promoter), 6 μL of annealed sgRNA, and 1 μL of T4 DNA ligase were added sequentially. Sterile double-distilled water was added to a final volume of 10 μL, and the mixture was incubated at 25 ℃ for 10 min before transformation.

[0058] The ligation product was transformed into DH5α E. coli competent cells and plated on TSA / Amp resistant plates. Single colonies were picked, amplified, and sequenced. Knockout plasmids Lenti-sg1-TMEM214 and Lenti-sg2-TMEM214 with correct sequences were screened out. The above plasmids were extracted and purified using an endotoxin-free plasmid extraction kit, and their concentrations were determined.

[0059] (5) 293T cells were cultured at a rate of 0.7 × 10⁻⁶. 6Cells were seeded at a density of 1 cell / mL in 2% FBS antibiotic-free DMEM medium. Transfection began when confluence reached 80%. The medium was replaced with fresh 2% FBS antibiotic-free DMEM before transfection. The transfection system was prepared according to the jetPrime transfection reagent instructions and included: 1.1 µg packaging plasmid psPAX2, 0.55 µg envelope plasmid pMD2.G, and 0.85 µg each of the target plasmids Lenti-sg1-TMEM214 and Lenti-sg2-TMEM214. 48 h after transfection, the cell culture supernatant was collected, and cell debris was removed using a 0.45 μm filter. The collected lentivirus packaging solution was stored at -80°C for later use.

[0060] (9) The generated virus solution was mixed at a volume ratio of 1:1 and transfected into RAW264.7-Cas9. After 48 hours of transfection, the medium was replaced with complete medium containing 0.5 μg / mL puromycin for positive polyclonal selection. The medium was changed every 48 hours. After all the untransfected control group cells died, the surviving positive polyclonal cells were expanded to a confluence of 75%–85%. Single cells were sorted into 96-well plates using flow cytometry and cultured statically for 24 hours. The medium was then replaced with complete medium containing 0.5 μg / mL puromycin. The growth of single clones was observed and marked daily. Well-grown positive single clone cell lines were selected for stepwise expansion. Proteins were extracted and Western blot detected using the same method as in Example 1. The results are as follows. Figure 2 As shown, the relative expression level of TMEM214 in TMEM214 KO was significantly reduced, indicating that the TMEM214 gene knockout cell line was successfully constructed.

[0061] Example 3: Application of TMEM214 gene / protein as a target in combating Brucella intracellular survival

[0062] In this embodiment, the TMEM214 gene / protein monoclonal knockout cell line obtained in Example 2 was infected with Brucella abortus strain A19 at 4, 24, 48, and 72 h. An uninfected TMEM214 gene / protein monoclonal knockout cell line was used as the control group. Intracellular viable bacterial counts were performed on both the control group and the Brucella-infected experimental group. The specific procedures are as follows:

[0063] (1) Control group RAW264.7 and TMEM214 KO cells were administered at 3×10 5 Seeds were inoculated per well in a 24-well plate with 1 mL of 2% serum-free DMEM medium without antibiotics.

[0064] (2) Brucella abortus strain A19 was used to infect control RAW264.7 and TMEM214 KO cells at a ratio of MOI=100.

[0065] (3) One hour after infection, 1 µL of 50 µg / mL gentamicin was added to each well for sterilization. Cell samples were collected at 0 h, 4 h, 24 h, 48 h and 72 h after infection.

[0066] (4) After centrifugation at 1100 rpm for 5 min, discard the supernatant, add 500 µL of 0.1% Triton to resuspend and pipette, and after 1 min at room temperature, perform serial dilution and spot count to detect the invasion level and intracellular survival of Brucella. Repeat three times for each group and each time point.

[0067] The results showed that in RAW264.7 cells, knocking out the TMEM214 gene significantly improved the intracellular survival level of Brucella abortus strain A19 at 24 h and 48 h. Figure 3 This indicates that the TMEM214 gene can significantly reduce the intracellular survival of Brucella bovis.

[0068] Example 4: Construction of TMEM214 gene / protein knockout mice and homozygous knockout mice

[0069] (1) For the mouse TMEM214 gene, exons 3 to 17 were selected as the target excision region. Specific Cas9 / gRNA binding target sites gRNA1 and gRNA2 were designed in the upstream flanking region of exon 3 and the downstream flanking region of exon 17, respectively.

[0070] gRNA1: 5'-CATGGTGAATTGTAAGAGGA AGG-3' (SEQ ID NO:9)

[0071] gRNA2: 5'-TGAGAGACCTCACAGGCCTG TGG-3' (SEQ ID NO: 10)

[0072] Primers guided by the Cas9 protein

[0073] Primer I: 5'-TCAGCTCCACAGAAGCAGTG-3' (SEQ ID NO:11).

[0074] Primer II: 5'-AGTCACCTAGGCTGCTGGTA-3' (SEQ ID NO: 12),

[0075] Primer III: 5'-CTCTCGTCTCCTGTCTTGCC-3' (SEQ ID NO:13).

[0076] Primer IV: 5'-CCCAAGGAGACCCAGGACTA-3' (SEQ ID NO:14) was used to cut the end of the gene, precisely removing a large effective coding region of 5580 bp, thereby achieving functional knockout of the TMEM214 gene.

[0077] (2) Preparation of F0 generation chimeric mice by microinjection of fertilized eggs:

[0078] A specific gRNA fragment targeting the mouse TMEM214 gene was synthesized in vitro and assembled with the Cas9 proteome to form a ribonucleoprotein complex (RNP). Female mice with a C57BL / 6JCya background were subjected to superovulation treatment, and fertilized eggs were collected. The prepared RNP complex was co-injected into the fertilized eggs, and the well-developed fertilized eggs were subsequently transferred into the oviducts of pseudopregnant surrogate female mice to breed offspring.

[0079] (3) Genotyping of offspring mice: After the surrogate mother mice gave birth to newborn mice, tail tissue of the pups was collected and genomic DNA was extracted. Specific identification primers were designed around the deletion region for PCR amplification and sequencing analysis to screen for positive F0 generation mice with a large deletion of the TMEM214 gene (successful exon 3-17 excision).

[0080] Positive F0 mice were crossbred with wild-type C57BL / 6JCya mice for passage purification. Specific forward primer P1 and reverse primer P2 were designed around the periphery of large genomic deletion regions, while a reverse primer P4 was designed inside the deletion regions. Primer sequences and types are as follows:

[0081] P1 (Forward): 5'-TCAGCTCCACAGAAGCAGTG-3' (SEQ ID NO: 11);

[0082] P2 (Reverse): 5'-AGTCACCTAGGCTGCTGGTA-3' (SEQ ID NO: 12);

[0083] P4 (Reverse): 5'-GGGGTTCCCAGTGAACACGCT-3' (SEQ ID NO: 15).

[0084] PCR typing is determined as follows:

[0085] Wild type (WT, + / +): Only a 318 bp band was observed;

[0086] Heterozygous mice (+ / -): both 318 bp and 468 bp bands were observed simultaneously;

[0087] Homozygous knockout (KO, - / -): Only a 468 bp band appears.

[0088] The results are as follows Figure 4 As shown, the TMEM214 knockout homozygous mouse model was successfully constructed.

[0089] Example 5: Application of TMEM214 gene / protein as a target in the treatment of brucellosis

[0090] (1) Grouping and infection treatment of experimental animals: 6-8 week old TMEM214 gene knockout homozygous mice with good developmental status, constructed and successfully identified in Example 4 of this invention, were selected as the experimental group. Wild-type (WT) C57BL / 6JCya mice of the same age and genetic background were selected as the control group. Mice in the experimental and control groups were administered 1×10⁻⁶ doses per mouse. 6 Intraperitoneal injection of standard doses of CFU of bovine Brucella abortus strain A19. Following inoculation, the bacteria were continuously observed and maintained under the same external environmental and feeding conditions for two weeks.

[0091] (2) Spleen sample collection from mice: Two weeks after infection, mice in the experimental and control groups were euthanized painlessly. Under aseptic conditions, the spleens of each group of mice were immediately dissected and completely removed. The wet weight of each spleen was measured in a sterile weighing dish and recorded. Subsequently, the spleen samples from each group were placed in tissue homogenization tubes containing 1 mL of sterile PBS.

[0092] (3) Detection and counting of bacterial load in spleen (CFU): Spleen samples from each group were thoroughly homogenized using a micro-tissue homogenizer to obtain a homogenized spleen tissue homogenate. 100 μL of the initial homogenate was taken and serially diluted 10-fold using sterile PBS. 50 μL of each dilution was evenly spread onto TSA solid medium plates. The plates were incubated upside down in a 37°C incubator for 3-4 days. After single colonies appeared, accurate colony counting was performed on plates with colony counts between 30 and 300.

[0093] Experimental results ( Figure 5 The study showed that, compared to wild-type control mice, homozygous TMEM214 knockout mice exhibited a significant increase in spleen bacterial load. This experiment demonstrates that TMEM214 gene / protein expression can significantly inhibit the intracellular proliferation and parasitism of Brucella in mammals, clearing intracellular Brucella. Therefore, the TMEM214 gene / protein could serve as an effective target for screening or developing therapeutic agents (synergists, activators) for brucellosis.

[0094] As can be seen from the above embodiments, the present invention provides the application of the TMEM214 gene as a target in the preparation of drugs for treating Brucella infection, providing a novel target for the development of anti-brucellosis targeted drugs and new vaccines.

[0095] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. TMEM214 Genes and / or TMEM214 The application of gene-encoded proteins as targets in the preparation of drugs for the prevention and / or treatment of brucellosis; TMEM214 The nucleotide sequence of the gene is shown in SEQ ID NO:

1. TMEM214 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:

2.

2. The application according to claim 1, characterized in that, The ability to promote TMEM214 protein or TMEM214 Gene expression.

3. The application according to claim 1, characterized in that, Brucella is Brucella bovis ( B. abortus )A19 strain.

4. A drug for treating brucellosis, characterized in that, Includes the ability to increase TMEM214 Active ingredients that express or enhance the activity of TMEM214 protein, as well as pharmaceutically acceptable carriers, excipients, or adjuvants.

5. The drug according to claim 4, characterized in that, The active ingredient of the drug is TMEM214 Any one or more of the following: gene overexpression vector, TMEM214 recombinant protein, and TMEM214 protein agonist.

6. A kind TMEM214 The gene knockout RAW264.7 cell line is characterized by, Targeted knockout using the CRISPR-Cas9 system TMEM214 The sgRNA target sequence is constructed from the 5' end sequence of the gene open reading frame, and the target sequence is shown in SEQ ID NO:3 and SEQ ID NO:

6.

7. A kind TMEM214 The homozygous knockout mouse model is characterized by, mice TMEM214 Knockout of exons 3 through 17 of the gene using Cas9 / gRNA technology resulted in the loss or functional inactivation of the TMEM214 protein; compared with wild-type mice, homozygous knockout mice showed a significant increase in splenic bacterial load after infection with Brucella.

8. The method according to claim 7 TMEM214 The homozygous knockout mouse model is characterized by, The Cas9 / gRNA binding target site sequence is shown in SEQ ID NO:9 and SEQ ID NO:

10.

9. The claim 6 TMEM214 Gene knockout RAW264.7 cell line or as described in claim 7 TMEM214 Application of homozygous knockout mouse models in studying the mechanism of Brucella intracellular infection and screening anti-brucellosis drugs.

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