Application of ABHD13 gene or protein in regulating avian innate immune response

Abstract

This invention belongs to the field of genetic engineering and discloses the application of the ABHD13 gene or protein in regulating the innate immune response in birds. This invention reveals for the first time the use of the ABHD13 gene or protein in regulating the innate immune pathway, elucidating from this perspective the mechanism by which the depalmitase ABHD13 regulates the innate immune pathway receptor adaptor protein NOD1, providing strong scientific evidence and tools for the prevention, control, and treatment of Gram-negative bacteria such as Salmonella. The oligonucleotides targeting the ABHD13 gene provided by this invention can significantly enhance the phagocytic capacity of chicken macrophages, providing a scientific means for scientific research and industrial applications related to the control of Gram-negative bacteria such as Salmonella.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically, it relates to the application of the ABHD13 gene or protein in regulating the innate immune response in birds. Background Technology

[0002] Salmonella is a group of Gram-negative bacteria that can infect multiple hosts. It is relatively anaerobic, non-spore-producing, and belongs to the Enterobacteriaceae family. These are rod-shaped intracellular facultative pathogens, ranging in size from 2 to 3 micrometers, possessing pili, and generally lacking capsules and spores. There are over 2000 serotypes. Salmonella can survive under various conditions. Its ability to adapt to host biological conditions and the resulting pathogenicity depend on the serotype of the Salmonella genus (Fàbrega et al., 2013; McSorley et al., 2014).

[0003] Avian Salmonella is transmitted both vertically and horizontally, causing acute or chronic diseases in poultry. It infects healthy poultry via the digestive tract, resulting in high morbidity and mortality rates, making it one of the most serious diseases affecting poultry farming. Pullorum disease, caused by Salmonella pullorum, occurs in chickens of all breeds and ages, but is most severe in chicks under three weeks old, with high mortality rates. In adult chickens, it is often chronic, significantly impacting egg production (Zhang Shengguo et al., 2023). Fowl typhoid, caused by Salmonella typhi, primarily affects adult chickens, characterized by enlarged liver and spleen, with the liver appearing yellowish-green or bronze (Hu Wenting, 2021).

[0004] In poultry production, Salmonella typhimurium (Salmonella typhimurium) Salmonella Typhimurium Salmonella enteritidis (ST) can not only cause illness and even death in poultry, but also contaminate poultry products, carrying Salmonella enteritidis and thus endangering human health. Most human infections with Salmonella are related to the consumption of poultry-derived foods such as eggs and chicken. The public health crisis caused by Salmonella infection in poultry has brought huge economic losses to the poultry industry and has become a major problem plaguing the industry (Jiang Yongjun, 2014; Xu Chenggang et al., 2015). Summary of the Invention

[0005] The purpose of this invention is to provide the application of the ABHD13 gene or protein in regulating the innate immune response in birds.

[0006] In order to achieve the purpose of this invention, in a first aspect, this invention provides the application of the ABHD13 gene or protein in regulating the innate immune response in birds (including non-disease diagnosis and treatment purposes).

[0007] In this invention, the reference sequence number of the ABHD13 gene from chicken on NCBI is NM_001008681.3.

[0008] The innate immune response in poultry refers to the immune response initiated by chickens upon sensing Gram-negative bacterial infection.

[0009] Preferably, the Gram-negative bacterium described in this invention is Salmonella ( Salmonella Shigella ( Shigella ), and more preferably Salmonella typhimurium ( Salmonella Typhimurium ).

[0010] Specifically, when the expression level of the ABHD13 gene or protein increases, the innate immune response in birds decreases; when the expression level of the ABHD13 gene or protein decreases, the innate immune response in birds increases and the phagocytic capacity of the chicken mononuclear macrophage cell line (HD11) is enhanced.

[0011] Secondly, this invention provides the application of the ABHD13 gene as a target gene in improving the innate immune response of poultry (including non-disease diagnosis and treatment purposes).

[0012] Preferably, the poultry mentioned in this invention is a chicken.

[0013] Thirdly, this invention provides the application of ABHD13 gene or protein inhibitors in the preparation of formulations that enhance the innate immune response of poultry (including non-disease diagnosis and treatment purposes).

[0014] Fourthly, the present invention provides the use of ABHD13 gene or protein inhibitors in the preparation of formulations for the prevention and / or protection against infection of poultry with Gram-negative bacteria.

[0015] The inhibitor can be selected from small molecule inhibitors, oligonucleotides, antibodies, peptides, or fusion proteins, etc.

[0016] Preferably, the oligonucleotide is miRNA, sgRNA, siRNA, shRNA, dsRNA, cDNA, or antisense RNA / DNA, more preferably shRNA targeting the ABHD13 gene.

[0017] More preferably, the nucleotide sequence of the shRNA targeting the ABHD13 gene is shown in SEQ ID NO:1.

[0018] Fifthly, the present invention provides an oligonucleotide, the sequence of which is shown in SEQ ID NO:1.

[0019] In a sixth aspect, the present invention provides a drug or composition for preventing and / or treating Gram-negative bacterial infections and enhancing the innate immune response of poultry, wherein the active ingredient is an ABHD13 gene or protein inhibitor.

[0020] Preferably, the poultry is a chicken.

[0021] Preferably, the Gram-negative bacteria are Salmonella, Shigella, etc., and more preferably Salmonella typhimurium.

[0022] Preferably, the active ingredient of the drug or composition is the oligonucleotide shown in SEQ ID NO:1.

[0023] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: (i) This invention discloses the use of the ABHD13 gene or protein in regulating the innate immune pathway. From this perspective, it elucidates the mechanism by which the depalmitase ABHD13 regulates the innate immune pathway receptor adaptor protein NOD1, providing a strong scientific basis and tool for the prevention, control and treatment of Gram-negative bacteria such as Salmonella.

[0024] (ii) The oligonucleotides targeting the ABHD13 gene provided by this invention can significantly improve the phagocytic capacity of chicken macrophages, providing a scientific means for scientific research and industrial application related to the prevention and control of Gram-negative bacteria such as Salmonella. Attached Figure Description

[0025] Figure 1 This represents the interference efficiency of shABHD13 detected by real-time PCR in Example 1 of this invention. shABHD13 can effectively reduce the expression of the ABHD13 gene. shABHD13 was the experimental group, and sh-NC was the control group. express P <0.05.

[0026] Figure 2 This invention relates to Example 1, which describes the detection of phagocytic capacity of chicken macrophages using flow cytometry. Knockdown of ABHD13 expression significantly reduced the phagocytic capacity of chicken macrophages. The sh-ABHD13 group was the experimental group, and the sh-NC group was the control group. express P <0.01.

[0027] Figure 3 This invention relates to Example 2, which describes the detection of the fluorescence intensity of the NF-κB reporter vector using a dual luciferase reporter assay. After treatment with Lipopolysaccharide, HD11 cells showed a significant decrease in NF-κB activity when the ABHD13 gene was overexpressed. The experimental group was flagABHD13, and the control group was flag. express P <0.01.

[0028] Figure 4The fluorescence intensity of the NF-κB reporter vector was detected by the dual luciferase reporter assay in Example 2 of this invention. After HD11 cells were treated with lipopolysaccharide, the activity of the transcription factor NF-κB increased significantly when the expression of the ABHD13 gene was interfered with. shABHD13 was the experimental group, and shNC was the control group. This indicates that P < 0.05. express P <0.01.

[0029] Figure 5 This invention provides an example of an immunoprecipitation combined with Western blot assay to identify the interaction between ABHD13 and NOD1 proteins. Detailed Implementation

[0030] This invention uses the chicken mononuclear macrophage cell line (HD11) as experimental material and employs flow cytometry (FCM) and affinity purification-mass spectrometry (AP-MS) to study the protein-protein interaction regulation of the chicken innate immune pathway. It was found that the protein ABHD13 can regulate the phagocytic capacity of chicken mononuclear macrophages and interacts with NOD1, the innate immune pattern recognition receptor protein that senses infection by Gram-negative bacteria such as Salmonella. When the expression level of ABHD13 gene or protein increases, the activity of NK-κB transcription factor decreases significantly. Conversely, when the expression level of ABHD13 gene or protein decreases, the activity of NK-κB transcription factor increases significantly and the phagocytic capacity of chicken mononuclear macrophages is enhanced.

[0031] ABHD13 is a depalmitase that can regulate the level or activity of certain signaling molecules, affecting signal transduction pathways, either directly or indirectly. The ABHD13 protein contains a FrsA domain with α / β hydrolase activity.

[0032] The present invention adopts the following technical solution: This invention provides an application of the ABHD13 gene or protein in regulating the phagocytic capacity and innate immune pathway activity of chicken mononuclear macrophages (HD11). First, a second-generation lentivirus was packaged using a plasmid targeting and interfering with ABHD13. The viral supernatant was collected, purified, concentrated, and then used to infect HD11 cells, obtaining a stable HD11 cell line with ABHD13 interference. When the phagocytic capacity of HD11 cells was evaluated using flow cytometry (FCM), the shRNA interference with ABHD13 protein expression resulted in a significantly lower HD11 positivity rate (approximately 8.6%) compared to the control group.

[0033] Based on this, the present invention provides an application of the ABHD13 gene or protein as a drug target in improving the innate immune response of chickens.

[0034] Furthermore, the present invention provides the application of an ABHD13 gene or protein inhibitor in the preparation of a drug that enhances antibacterial activity; and the application of an ABHD13 gene or protein inhibitor in the preparation of a drug for the prevention and / or treatment of Gram-negative bacteria such as Salmonella.

[0035] The term "application" as used in this invention can refer to applications for therapeutic purposes or applications for non-therapeutic purposes, such as scientific research.

[0036] The "drug" or "inhibitor" described in this invention may be selected from small molecule inhibitors, oligonucleotides, antibodies, peptides, or fusion proteins. The oligonucleotide is preferably miRNA, siRNA, or shRNA, and more preferably shRNA targeting the ABHD13 gene.

[0037] This invention provides a set of oligonucleotides, the sequences of which are shown in SEQ ID NO:1. These oligonucleotides can be used to prevent and / or treat infections caused by Gram-negative bacteria such as Salmonella and to enhance the innate immunity of chickens.

[0038] Based on this, the present invention provides a kit containing an oligonucleotide sequence as shown in SEQ ID NO:1.

[0039] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0040] Example 1: shRNA interferes with ABHD13 gene expression, reducing the phagocytic capacity of chicken mononuclear macrophages. 1. shRNA design and experimental grouping shRNA sequences were designed targeting the chicken ABHD13 gene sequence (Gene ID: 418763). Sequences SEQ ID NO:1 and SEQ ID NO:2 were synthesized by Beijing Qingke Biotechnology Co., Ltd. The shRNA sequences are shown in Table 1. The plasmid backbone used was pLV3ltr-ZsGreen-Puro-U6 (purchased from Beijing Qingke Biotechnology Co., Ltd.). shNC served as a negative control.

[0041] Table 1

[0042] The experiment was divided into two groups: the experimental group (shABHD13) and the control group (shNC).

[0043] 2. HEK293T cell culture (human embryonic kidney cells, purchased from the Cell Bank of the Chinese Academy of Sciences) (1) Cell resuscitation: Remove the frozen cells from liquid nitrogen, place them in a 37°C water bath and shake them rapidly. After the cell solution is completely dissolved, add the cell solution to 9 mL of complete culture medium and mix well. Centrifuge at 3000 rpm for 5 minutes, remove the supernatant, mix the cell pellet thoroughly with fresh complete culture medium, add it to a cell culture dish, mix well, and incubate in a 37°C, 5% CO2 incubator.

[0044] (2) Cell medium change: Observe cell density and morphology under a microscope. Generally, change the medium every 24 hours. Warm the cell culture medium in advance, discard the old culture medium, and add the new culture medium.

[0045] (3) Cell passage: Under a microscope, when the cell confluence reaches 70%-80%, passage should be performed. Discard the old culture medium, gently wash the cells with PBS, add 1.5 mL of 0.25% trypsin, and digest until the cells begin to suspend. Immediately add 4.5 mL of complete culture medium to stop the digestion and prepare a single-cell suspension. Add the cell suspension to a 15 mL centrifuge tube, centrifuge at 3000 rpm for 5 min, discard the supernatant, fully suspend the cell pellet with 6 mL of complete culture medium, add it to a cell culture dish, mix well, and incubate at 37℃ in a 5% CO2 incubator.

[0046] 3. shRNA plasmid lentiviral packaging The plasmid was transfected into HEK293T cells using liposome transfection.

[0047] Cell transfection was performed according to the Lipofectamine® 3000 reagent (Life Technologies) instructions: (1) Dilute Lipofectamine® 3000 reagent with Opti-MEM® medium and mix thoroughly. Prepare plasmid premix in a ratio of shRNA plasmid: packaging plasmid psPAX2: envelope plasmid pMD2.G=1:2:1 and dilute with Opti-MEM® medium. (2) Add P3000™ reagent to the plasmid premix. Add diluted plasmid premix (1:1 volume ratio) to each tube of diluted Lipofectamine® 3000 reagent. After incubating at room temperature for 5 min, transfer the plasmid-liposome complex to the cell culture dish. (3) 6-8 h after transfection, the medium was gently changed to virus harvesting medium. 48 h after transfection, the supernatant was collected, centrifuged at 400 g for 5 min, and filtered with a 0.45 µm filter (Millipore, Millex®-HP). The virus was concentrated using PEG-it™ (SBI, LV825A-1), and the virus titer was determined using a p24 ELISA (GenScript, L00938) detection kit.

[0048] 4. Lentiviral transfection and flow cytometry sorting (1) Chicken mononuclear macrophage cell line (HD11, kindly provided by Professor Chen Guohong of Yangzhou University) was cultured using the same method as the HEK293T culture method described above. (2) When the cell density is 60-70%, add Polybrene (MCE, HY-112735) to the culture medium at a final concentration of 8µg / ml for 4-6h. (3) Add the concentrated shRNA lentivirus to the cell culture medium, mix well, and infect for 48 hours; (4) After the infection is over, discard the old culture medium, rinse with PBS, add 0.25% trypsin (Gibco) and digest in a 37°C incubator for 5 minutes. Gently shake the culture dish, and when cells are seen to be free under a microscope, immediately add 2 times the volume of complete culture medium to stop the digestion. (5) Add the cell suspension to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, remove the supernatant, resuspend the cells in PBS and transfer them into a flow cytometry tube, and perform flow cytometry sorting based on the GFP fluorescence signal to obtain stably transfected positive cells.

[0049] 5. Quantitative Real-Time PCR Experiment RNA extraction was performed according to the instructions of the total RNA extraction kit (Tiangen, DP419). Reverse transcription was performed according to the FastQuantcDNA first-strand synthesis kit (Tiangen, KR106). The interference efficiency of shABHD13 was detected by real-time quantitative PCR. The qPCR reaction solution was prepared using the One Step SYBR® PrimeScript™ RT-PCR Kit II (Takara). The expression level of ABHD13 was analyzed using the ABI Q7 Flex system (Applied Biosystems, USA). β-Actin was used as an internal control gene. -ΔΔct Method calculation.

[0050] 6. Salmonella Typhimurium infection and flow cytometry analysis The shABHD13 and shNC groups were infected with Salmonella typhimurium with mCherry fluorescent tags. Three biological replicates were set up for each group. After 4 hours of infection, the culture medium was replaced with gentamicin at a concentration of 100 µg / ml and cultured for another 12 hours to kill extracellular bacteria. The cells were collected and made into a cell suspension with PBS and transferred into flow cytometry tubes for analysis of cell positivity rate.

[0051] 7. Results See Figure 1This study demonstrates the interference efficiency of quantitative real-time PCR in detecting shABHD13. shABHD13 was the experimental group, and shNC was the control group. β-Actin was used as the internal control gene, and 2... -ΔΔct The relative expression levels of ABHD13 and β-Actin were calculated. As shown in the figure, shABHD13 can effectively reduce the expression level of ABHD13.

[0052] See Figure 2 This study demonstrates the phagocytic capacity of chicken macrophages as detected by flow cytometry. shABHD13 was the experimental group, and shNC was the control group. As shown in the figure, knocking down the expression level of ABHD13 significantly reduced the Salmonella positivity rate in HD11 cells.

[0053] Example 2: Overexpression of the ABHD13 gene significantly reduced NK-κB transcription factor activity. 1. Experimental grouping and layout The experiment was divided into five groups: experimental group (Flag-ABHD13), control group (Flag), control group, promoter group, and basic group, with three biological replicates in each group. The exogenous expression Flag-ABHD13 and Flag transfected cell lines obtained in Example 1 and the wild-type HD11 cell line were seeded into 24-well plates according to the experimental design and cultured in a 37°C, 5% CO2 incubator.

[0054] 2. Transfection of HD11 cell lines expressing exogenous ABHD13 and LPS treatment When the seeded cell density is approximately 80%, cell transfection should be performed according to the TransIT-X2® Dynamic Delivery System reagent (Mirus, MIR6000) instructions. (1) Dilute the transferred luciferase reporter plasmid with Opti-MEM® medium and mix thoroughly to prepare a plasmid premix solution; (2) Add TransIT-X2 reagent to the plasmid premix according to the recommended ratio, mix thoroughly, incubate at room temperature for 15 min, and then add the complex to the cell culture dish; (3) 24 h after transfection, LPS (lipopolysaccharide, purchased from Sigma) was added to the culture dish to a final concentration of 1 µM and the cells were treated for 6 h. At the end of the treatment, luciferase chemiluminescence was detected.

[0055] 3. Dual-luciferase reporter gene assay Dual-Luciferase reporter gene assay was performed according to the instructions for the Dual-Luciferase® Reporter Assay System (Promega, E1910): (1) Cell lysis: Remove the culture medium, add cell lysis buffer to the cell culture plate, and lyse thoroughly. 4 ° C, centrifuge at 12000 rpm for 5 min, and collect the supernatant for subsequent determination; (2) Preparation of Renali luciferase detection buffer: Calculate the required Renali luciferase detection buffer according to the volume of 100 µL for each sample, and prepare the working solution according to the ratio of Renali luciferase detection substrate: Renali luciferase detection buffer = 1:100. (3) Determination of firefly luciferase: Add 100 µL of cell lysis buffer to the microplate, then add 100 µL of firefly luciferase detection reagent, mix well by pipetting, place in the microplate reader, and the value of chemiluminescence is the firefly luciferase value. (4) Determination of Renal luciferase: After the above steps are completed, add 100 µL of Renal luciferase detection buffer, mix by pipetting, mix by the above method, and place in an ELISA reader to determine the Renal luciferase value. (5) Calculation: The ratio of the luciferase value of firefly to that of sea urchin is the activity of the relevant reporter gene.

[0056] 4. Results See Figure 3 The study demonstrated that after LPS treatment, when the expression of the ABHD13 gene was interfered with, the chemiluminescence intensity of HD11 cells showed a significant increase in the activity of the transcription factor NF-κB, indicating that ABHD13 regulates the expression of downstream immune factors by affecting the activity of the transcription factor NF-κB.

[0057] Example 3: Exogenous overexpression of ABHD13 gene significantly reduced NK-κB transcription factor activity. 1. Construction of overexpression vectors Based on the pcDNA3.1-3×Flag C vector information (see Master's thesis, Wang Fei, Molecular mechanism of SPOP regulating innate immunity through negative feedback degradation of MyD88, DOI: 10.27630 / d.cnki.gznky.2020.000009) and the chicken ABHD13 gene sequence published on NCBI (GenBank: NM_001008681.3), the gene CDS sequence was synthesized by Beijing Qingke Biotechnology Co., Ltd. and ligated into the vector.

[0058] 2. Experimental grouping and layout The experiment was divided into five groups: experimental group (FLAG-ABHD13), control group (FLAG), control group, promoter group, and basic group, with three biological replicates in each group. The FLAG-ABHD13 and FLAG stably transfected cell lines obtained in Example 3, along with the wild-type HD11 cell line, were seeded into 24-well plates according to the experimental design and cultured at 37°C in a 5% CO2 incubator.

[0059] 3. HD11 cell line transfected with exogenous overexpression of ABHD13 and treated with LPS When the seeded cell density is approximately 80%, cell transfection should be performed according to the TransIT-X2® Dynamic Delivery System reagent (Mirus, MIR6000) instructions. (1) Dilute the transferred luciferase reporter plasmid with Opti-MEM® medium and mix thoroughly to prepare a plasmid premix solution; (2) Add TransIT-X2 reagent to the plasmid premix according to the recommended ratio, mix thoroughly, incubate at room temperature for 15 min, and then add the complex to the cell culture dish; (3) 24 h after transfection, LPS (lipopolysaccharide, purchased from Sigma) was added to the culture dish to a final concentration of 1 µM and the cells were treated for 6 h. At the end of the treatment, luciferase chemiluminescence was detected.

[0060] 3. Dual-luciferase reporter gene assay Dual-Luciferase reporter gene assay was performed according to the instructions for the Dual-Luciferase® Reporter Assay System (Promega, E1910): (1) Cell lysis: Remove the culture medium, add cell lysis buffer to the cell culture plate, and lyse thoroughly. 4 ° C, centrifuge at 12000 rpm for 5 min, and collect the supernatant for subsequent determination; (2) Preparation of Renali luciferase detection buffer: Calculate the required Renali luciferase detection buffer according to the volume of 100 µL for each sample, and prepare the working solution according to the ratio of Renali luciferase detection substrate: Renali luciferase detection buffer = 1:100. (3) Determination of firefly luciferase: Add 100 µL of cell lysis buffer to the microplate, then add 100 µL of firefly luciferase detection reagent, mix well by pipetting, place in the microplate reader, and the value of chemiluminescence is the firefly luciferase value. (4) Determination of Renal luciferase: After the above steps are completed, add 100 µL of Renal luciferase detection buffer, mix by pipetting, mix by the above method, and place in an ELISA reader to determine the Renal luciferase value. (5) Calculation: The ratio of the luciferase value of firefly to that of sea urchin is the activity of the relevant reporter gene.

[0061] 4. Results See Figure 4 The study demonstrated that after LPS treatment, when the ABHD13 gene was overexpressed in HD11 cells, the chemiluminescence intensity showed a significant decrease in the activity of the transcription factor NF-κB, indicating that ABHD13 regulates the expression of downstream immune factors by affecting the activity of the transcription factor NF-κB.

[0062] Example 4: Verification of the interaction between protein ABHD13 and protein NOD1 1. Construction of ABHD13 and NOD1 gene expression vectors Based on the pcDNA3.1-3×Flag C vector information (see Master's thesis, Wang Fei, Molecular mechanism of SPOP regulating innate immunity through negative feedback degradation of MyD88, DOI: 10.27630 / d.cnki.gznky.2020.000009) and the chicken ABHD13 gene sequence published on NCBI (GenBank: NM_001008681.3), the gene CDS sequence was synthesized by Beijing Qingke Biotechnology Co., Ltd. and ligated into the vector.

[0063] Based on the pcDNA3.1-GFP vector information and the chicken NOD1 gene sequence published on NCBI (GenBank: NM_001318438.1), the gene CDS sequence was synthesized by Beijing Qingke Biotechnology Co., Ltd. and ligated into the vector.

[0064] 2. DF1 cell culture The culture method for chicken embryo fibroblasts (DF1, purchased from the Cell Bank of the Chinese Academy of Sciences) was the same as that for HEK293T cells described above.

[0065] 3. Cell transfection When the cells reached 80% confluence, the gene expression vectors ABHD13-pcDNA3.1-3×Flag C and NOD1-pcDNA3.1-GFP were transfected into DF1 cells using liposome transfection. The transfection reagent used was Lipofectamine® 3000 (Life Technologies), and the procedure was the same as described above. Cells were collected 24 hours after transfection for downstream experiments.

[0066] 4. Protein immunoprecipitation (1) Pretreatment of magnetic beads: A / G protein affinity beads and FLAG tag affinity beads were washed four times with protein extraction buffer (RIPA Buffer), each time for 5 minutes. The washing process was carried out on a shaker at 4°C and required slow shaking.

[0067] (2) Cell collection and lysis: Add 5 times the amount of RIPA Buffer to the cells obtained in step 3 to lyse the cells, 4 ° Centrifuge at 12000 rpm for 30 min. Collect the supernatant and place it on ice for later use.

[0068] (3) Preclearing background proteins: Add 20 μL of pretreated A / G protein magnetic beads to the lysis buffer and shake slowly on a shaker at 4°C for 2 h to remove non-specific contaminating proteins and reduce background. After centrifugation, collect the supernatant into a new centrifuge tube. Take 1 / 50 of the lysis buffer (total protein) and store it at -80°C as a positive control (Input). The remaining lysis buffer is used for immunoprecipitation (IP), i.e., the experimental group.

[0069] (4) IP: Add FLAG-labeled magnetic beads to the lysis solution used as IP and shake slowly overnight on a shaker at 4°C.

[0070] (5) Washing: Centrifuge to collect agarose beads, discard the supernatant, and wash four times with protein extraction buffer at 4°C on a shaker. The first two washes are for 5 min each, and the last two washes are for 30 min each. Wash twice with 54K buffer, the first wash is for 5 min, and the second wash is for 30 min.

[0071] (6) Purification: Add 60µL TBS solution and 5µL FLAG-tagged peptide, shake at 4℃ for 1h, and centrifuge to extract the supernatant.

[0072] (7) Add sample loading buffer, boil at 96°C for 10 min, immediately place on ice, centrifuge briefly, and then aspirate the supernatant for polyacrylamide gel electrophoresis.

[0073] 5. Western blot (1) The protein was transferred to a PVDF membrane (Millipore) after polyacrylamide gel electrophoresis. The transfer conditions were 350mA constant current.

[0074] (2) After blocking the PVDF membrane for 30 min, incubate with primary antibody for 1 h, wash the membrane three times with TBST for 5 min each time, then incubate with secondary antibody, wash the membrane three times with TBST for 5 min each time.

[0075] (3) Use the ImageQuant LAS 4000mini instrument for automatic exposure to acquire Western hybridization images.

[0076] The primary antibody used in this experiment was a DDDDK tag antibody (Anti-DDDDK tag antibody, AE005, Aibotek), the primary antibody used for the GFP tag was a GFP tag antibody (Anti-GFP tag antibody, AE078, Aibotek), and the secondary antibody was HRP-labeled affinity-purified goat anti-rabbit IgG (H+L) (AS014, Aibotek).

[0077] 6. Results See Figure 5This study demonstrates the identification of the interaction between ABHD13 and NOD1 proteins using a combination of immunoprecipitation and Western blot assays. The interaction verification experiment consisted of three groups: lane 1 was used for co-transfection of pcDNA3.1-GFP and the ABHD13 gene expression vector; lane 2 was used for co-transfection of pcDNA3.1-Flag and the NOD1 gene expression vector; and lane 3 was used for co-transfection of the ABHD13 and NOD1 gene expression vectors. Cells were collected after 24 hours, and the FLAG-tagged fusion protein was purified by immunoprecipitation. Anti-FLAG Western blot analysis showed that the FLAG-tagged ABHD13 protein was successfully enriched, indicating successful expression of the ABHD13 gene expression vector in DF1 cells. Anti-GFP Western blot analysis showed that the GFP-tagged NOD1 protein was present in the interacting protein complex, indicating an interaction between ABHD13 and NOD1.

[0078] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

[0079] References: [1]. Anna Fàbrega , Jordi Vila. Salmonella enterica serovar Typhimuriumskills to succeed in the host: virulence and regulation; Clin Microbiol Rev; 2013.4;26(2):308-41. doi: 10.1128 / CMR.00066-12. [2].Stephen J McSorley.Immunity to intestinal pathogens: lessons learned from Salmonella;Immunol Rev.2014.7;260(1):168-82. doi: 10.1111 / imr.12184. [3]. Zhang Shengguo, Yang Li, Zhang Yuan. Transmission routes and control measures of fowl tic. Modern Rural Science and Technology. 2023(06).

[0080] [4]. Hu Wenting. Prevention and control of salmonellosis in chickens. Poultry Science. 2021 (02).

[0081] [5]. Jiang Yongjun. Isolation, identification and drug susceptibility test of Salmonella Typhimurium in broilers. Chinese Journal of Animal Husbandry and Veterinary Medicine. 2014, 30 (11).

[0082] [6]. Xu Chenggang, Liao Xiaoping. Serotypes, drug resistance and PFGE type analysis of foodborne and human Salmonella in Guangdong Province from 2007 to 2014. Chinese Journal of Agricultural Science, 2015, 48(15): 3099-3108, DOI: 10.3864 / j.issn.0578-1752.2015.15.019.

[0083] [7]. Zhou, P., Y. She, et al Alpha-kinase 1 is a cytosolic innateimmune receptor for bacterial ADP-heptose. Nature, 2018. 561(7721): p. 122-126. [8]. Wang Fei. Molecular mechanism of SPOP regulating innate immunity through negative feedback degradation of MyD88 [D]. Chinese Academy of Agricultural Sciences, 2021. DOI: 10.27630 / d.cnki.gznky.2020.000009.

Claims

1. Application of ABHD13 gene or protein inhibitors in the preparation of formulations that enhance the innate immune response of poultry; wherein The poultry in question is chicken; The innate immune response capacity of poultry refers to the ability of chickens to sense Gram-negative bacterial infection and initiate an immune response. The gram-negative bacteria is Salmonella typhimurium Salmonella Typhimurium ); The reference sequence number of the ABHD13 gene from chicken on NCBI is NM_001008681.

3.

2. Application of ABHD13 gene or protein inhibitors in the preparation of formulations for the prevention and / or protection against Gram-negative bacterial infections in poultry; wherein The poultry in question is chicken; The reference sequence number of the ABHD13 gene from chicken on NCBI is NM_001008681.

3.

3. Use according to claim 1 or 2, characterized in that, The inhibitor is selected from small molecule inhibitors, oligonucleotides, antibodies, peptides, or fusion proteins; The oligonucleotide is miRNA, sgRNA, siRNA, shRNA, or dsRNA.

4. Use according to claim 3, characterized in that, The inhibitor is an shRNA that targets the ABHD13 gene, and its nucleotide sequence is shown in SEQ ID NO:

1.

5. An oligonucleotide, characterized in that, The sequence of the oligonucleotide is shown in SEQ ID NO:

1.

6. A drug or composition for preventing and / or treating Gram-negative bacterial infections and enhancing the innate immune response in poultry, characterized in that, The active ingredient is shRNA that targets the ABHD13 gene, and its nucleotide sequence is shown in SEQ ID NO:1; The poultry mentioned is chicken; The Gram-negative bacteria mentioned are Salmonella Typhimurium (Salmonella Typhimurium) Salmonella Typhimurium ); The reference sequence number of the ABHD13 gene from chicken on NCBI is NM_001008681.3.

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