Application of SIRT1 gene in regulating innate immune response of poultry

Abstract

The invention discloses an application of an SIRT1 gene in regulating and controlling innate immune response of poultry. The application of the SIRT1 gene or protein in regulating the innate immune pathway of poultry is disclosed for the first time, and powerful scientific basis and tools are provided for prevention, control and treatment of gram-negative bacteria such as salmonella. The inhibitor of the targeted gene SIRT1 provided by the invention can significantly improve the resistance of chicken macrophages to salmonella, and provides a scientific means for scientific research and industrial application related to prevention and control of gram-negative bacteria such as salmonella.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular, relates to the application of SIRT1 gene in regulating the innate immune response of birds. Background Art

[0002] Salmonella belongs to the family Enterobacteriaceae, a type of Gram-negative bacteria with a complex antigenic structure. It can infect a variety of hosts, is relatively anaerobic, does not produce spores, and is an intracellular facultative pathogen. It is approximately 0.7 to 1.5 μm in diameter and 2 to 5 μm in length. It has pili but generally does not have capsules or spores. There are more than 2,500 serotypes, which can seriously damage the intestinal health of the host after infection (FÀBREGA and VILA, 2013).

[0003] Avian Salmonella is transmitted vertically and horizontally. Poultry infected with Salmonella can cause acute or chronic diseases, with high mortality rates, causing huge economic losses to the poultry industry and becoming a major problem plaguing the poultry industry (Wang Min, 2009; SHAH et al., 2012). Common diseases caused by Salmonella in chickens include pullorum, fowl typhoid and avian paratyphoid. Pullorum mainly infects seven-day-old chicks, with a high mortality rate. Infection in adult chickens affects their fertilization rate and egg-laying performance. Typhoid mostly occurs in adult chickens and young chickens, and is acute or chronic. The symptoms are similar to pullorum, with enlarged liver and spleen, and the liver is yellow-green or bronze as the main characteristics. Avian paratyphoid is caused by Salmonella with flagella that can move, which can cause edema of the intestinal mucosa and local congestion. These three salmonellosis are the main bacterial diseases in chicken farms, which can cause large-scale deaths of chicks, reduce egg production and hatching rate of laying hens, and reduce the rate of healthy chicks, affecting the growth and development of chickens.

[0004] There is a close connection between poultry products (such as eggs and chicken) contaminated with Salmonella and human infection with Salmonella. For example, while Salmonella typhi and Salmonella enterica are common and highly infectious in poultry while spreading among humans, they are recognized by the World Health Organization as serious zoonoses and have received widespread attention from the public (VAN IMMERSEEL et al., 2005). Summary of the invention

[0005] The purpose of the present invention is to provide application of SIRT1 gene in regulating avian innate immune response.

[0006] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides the use of SIRT1 gene in regulating the innate immune response of avian animals (including non-disease diagnosis and treatment purposes).

[0007] In the present invention, the poultry is chicken.

[0008] The reference sequence number of the chicken SIRT1 gene on NCBI is NM_001004767.2.

[0009] SIRT1 is an NAD (nicotinamide adenine dinucleotide)-dependent histone deacetylase that is involved in regulating metabolism, aging, cell survival, autophagy, etc. SIRT1 activates multiple proteins and transcription factors such as PGC-1a and HIF-1a through deacetylation, promotes mitochondrial biogenesis, and is involved in many physiological and pathological processes, such as oxidative stress and cell apoptosis.

[0010] The avian innate immune response refers to the chicken's perception of Gram-negative bacterial infection and the initiation of an immune response.

[0011] Furthermore, the regulation refers to inhibiting the expression of SIRT1 gene at the transcriptional or translational level to enhance the resistance of chicken mononuclear macrophages to Salmonella.

[0012] Furthermore, the regulation refers to inhibiting the expression of SIRT1 gene at the transcriptional or translational level to enhance the innate immune response capability of avian animals.

[0013] In a second aspect, the present invention provides the use of a SIRT1 gene or protein inhibitor in the preparation of a preparation for improving the innate immune response ability of poultry.

[0014] In a third aspect, the present invention provides use of a SIRT1 gene or protein inhibitor in the preparation of a preparation for preventing and / or preventing poultry from being infected with Gram-negative bacteria.

[0015] The inhibitor may be selected from small molecule inhibitors, oligonucleotides, antibodies, polypeptides or fusion proteins.

[0016] Preferably, the inhibitor is Selisistat (EX527, SEN0014196), which has the following structure:

[0017] Furthermore, the Gram-negative bacteria is Salmonella.

[0018] In a fourth aspect, the present invention provides a medicine or composition for preventing and / or treating Gram-negative bacterial infection and improving the innate immune response ability of poultry, wherein the active ingredient is the above-mentioned SIRT1 gene or protein inhibitor.

[0019] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: (I) This invention reveals for the first time the use of SIRT1 gene or protein in regulating the innate immune pathway. From this perspective, it clarifies the mechanism of the deacetylase SIRT1 regulating the innate immune pathway, providing a strong scientific basis and tool for the prevention, control and treatment of Salmonella and other Gram-negative bacteria.

[0020] (ii) The inhibitor targeting the SIRT1 gene provided by the present invention enhances the resistance of chicken mononuclear macrophages to Salmonella, providing a scientific means for scientific research and industrial application related to the prevention and control of Salmonella and other Gram-negative bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The flow cytometry method was used to detect the anti-infection ability of chicken macrophages against Salmonella after treatment with SIRT1 Inhibitor or SIRT1 Activator in Example 1 of the present invention, where Inhibitor and Activator were experimental groups, and WT was a control group.

[0022] Figure 2 The expression of cellular immune factor IL-8 after Salmonella infection was detected by fluorescence quantitative PCR in Example 1 of the present invention. IL-8 was significantly upregulated after SIRT1 Inhibitor treatment and Salmonella infection, Inhibitor, ST and Inhibitor+ST were experimental groups, and Control was the control group.

[0023] Figure 3 Schematic diagram of the structure of the SIRT1 gene expression vector in Example 2 of the present invention.

[0024] Figure 4 The dual luciferase reporter assay for detecting NF-κB reporter in Example 2 of the present invention. The results of HD11 cells treated with LPS showed that overexpression of SIRT1 significantly reduced the activity of transcription factor NF-κB; on the contrary, the activity of transcription factor NF-κB significantly increased after Inhibitor treatment. FLAG-SIRT1 and Inhibitor were experimental groups, and FLAG and Control were control groups.

[0025] Figure 5 The expression of IL-1β and IL-8 after overexpression of SIRT1 or Inhibitor was detected by fluorescence quantitative PCR in Example 2 of the present invention. Overexpression of SIRT1 significantly reduced IL-1β; Inhibitor treatment significantly upregulated IL-1β and IL-8, FLAG-SIRT1 and Inhibitor were experimental groups, and FLAG and Control were control groups.

[0026] DETAILED DESCRIPTION

[0027] The present invention uses a chicken mononuclear macrophage cell line (HD11) as an experimental material and utilizes flow cytometry (FCM) to discover that the protein SIRT1 can regulate the ability of chicken mononuclear macrophages to resist Salmonella infection and regulate the activity of NK-κB transcription factors; when the expression level of SIRT1 gene or protein increases, the activity of NK-κB transcription factors significantly decreases; conversely, when the expression level of SIRT1 gene or protein decreases, the activity of NK-κB transcription factors significantly increases, and the resistance of chicken mononuclear macrophages to Salmonella is enhanced.

[0028] The present invention adopts the following technical solution: The present invention provides an application of a SIRT1 gene or protein in regulating the resistance of chicken mononuclear macrophages (HD11) to Salmonella and the activity of innate immune pathways. First, chicken macrophages are treated with a selective SIRT1 activator or inhibitor. When the ability of HD11 to resist Salmonella infection is evaluated by flow cytometry (FCM), the positive rate of HD11 after treatment with the SIRT1 activator is significantly higher than that of the control group by about 13.9%; the positive rate of HD11 after treatment with the inhibitor is significantly lower than that of the control group by about 15.35%.

[0029] On this basis, the present invention provides an application of a SIRT1 gene or protein as a drug target in improving the innate immune response ability of chickens.

[0030] Furthermore, the present invention provides an application of a SIRT1 gene or protein inhibitor in the preparation of a drug for enhancing antibacterial ability; and an application of a SIRT1 gene or protein inhibitor in the preparation of a drug for preventing and / or treating Salmonella and other Gram-negative bacteria.

[0031] The "application" mentioned in the present invention may refer to application for therapeutic purposes or application for non-therapeutic purposes, such as scientific research.

[0032] The "drug" or "inhibitor" of the present invention can be selected from small molecule inhibitors, oligonucleotides, antibodies, polypeptides or fusion proteins.

[0033] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present 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.

[0034] Example 1 Selective SIRT1 activator or inhibitor treatment of chicken macrophages, SIRT1 promotes Salmonella infection

[0035] 1. Cultivation of chicken mononuclear macrophage cell line (HD11, kindly donated by Professor Chen Guohong of Yangzhou University)

[0036] (1) Cell recovery: Take out the frozen cells from liquid nitrogen, quickly place them in a 37°C water bath and shake them continuously. After the cell solution in the cryopreservation tube is completely dissolved, add the cell suspension to 6 mL of preheated complete culture medium and mix well. Centrifuge at 1500 rpm for 5 minutes, remove the supernatant, mix the cell pellet thoroughly with new complete culture medium, add it to the cell culture bottle, shake well, and culture it in a 37°C, 5% CO2 incubator overnight.

[0037] (2) Cell culture medium replacement: Observe the cell density and morphology under a microscope. Generally, change the culture medium every 24 hours. Preheat the cell culture medium in advance, discard the old culture medium, and add new culture medium.

[0038] (3) Cell passaging: Observe under a microscope. When the cell confluence reaches about 80%-90% (the cell suspension turns yellow), the cell suspension is passaged. The cell suspension is transferred to a 15 ml centrifuge tube. PBS is added to a T25 cell culture flask (containing adherent cells) to gently wash the cells in the flask. The PBS is aspirated and 2 ml of 0.25% trypsin is added. When the cells change from irregular to round under a microscope, the cell and trypsin suspension is pipetted into the 15 ml centrifuge tube containing the cell suspension with a gun. The digestion is terminated and the tube is centrifuged at 1500 rpm for 5 min. The supernatant is discarded. The cells are fully resuspended with 1 ml of complete culture medium and the cell suspension is placed in new culture flasks (containing 5 ml of complete culture medium). The cells are shaken and cultured in a 37°C, 5% CO2 incubator.

[0039] 2. Selective SIRT1 activators or inhibitors

[0040] Selective SIRT1 Activator: SRT1720 HCL (Selleck.cn) is a selective SIRT1 activator. The molecular formula of SRT1720 HCL is as follows:

[0041] Selective SIRT1 inhibitor: Selisista (EX527, SEN0014196) (Selleck.cn) is a potent and selective SIRT1 inhibitor. The molecular formula of Selisista is as follows:

[0042] 3. Salmonella (Salmonella typhimurium, ST) infection and flow cytometry analysis

[0043] Experimental groups: experimental group (Activator; Inhibitor) and control group.

[0044] After treating the cells with Activator, 1uM; Inhibitor, 10uM for 24 hours, the experimental group and the control group were infected with Salmonella carrying mCherry fluorescent label, and 3 biological replicates were set for each group. After 4 hours of infection, the culture medium was changed to 100µg / ml gentamicin and cultured for 12 hours to kill the bacteria. The cells were collected and made into cell suspension with PBS and transferred to flow tube, and the cell positive rate was analyzed by flow cytometer.

[0045] 4. Inhibitor-treated cells infected with Salmonella, immune factor IL-8 was significantly upregulated

[0046] The method of treating chicken macrophage HD11 cells with inhibitor was the same as above. The cells were harvested 4 hours after Salmonella infection and RNA was extracted.

[0047] RNA extraction was performed according to the instructions of the total RNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., DP419), and reverse transcription was performed according to the FastQuantcDNA first-strand synthesis kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., KR106). The expression of cytokines after inhibitor treatment was detected by fluorescent quantitative PCR. The qPCR reaction solution was configured with the One Step SYBR® PrimeScript™ RT-PCR Kit II (Takara) and analyzed by the ABI Q7 Flex system (Applied Biosystems, USA). β-Actin was used as the internal reference gene, and 2 -ΔΔct Method calculation. The primers used for PCR are shown in Table 1.

[0048] Table 1

[0049] 5. Results

[0050] See also Figure 1 , showing the phagocytic ability of chicken macrophages detected by flow cytometry. WT is the control group, and Inhibitor and Activator are the experimental groups. As can be seen from the figure, the positive rate of Inhibitor treatment was significantly reduced, and the positive rate of Activator was significantly increased.

[0051] See also Figure 2, showing the expression of IL-8 in chicken macrophages treated with Inhibitor and infected with Salmonella (Salmonella typhimurium SL-1334-mCherry, i.e., Salmonella typhimurium SL1344 with fluorescent labeling) by fluorescence quantitative PCR. Inhibitor and Inhibitor+ST were experimental groups, and Control and ST were control groups. β-Actin was used as the internal reference gene, and 2 -ΔΔct Methods The relative expression of IL-8 and β-Actin was calculated. As shown in the figure, SIRT1 inhibitor treatment can significantly promote the expression of inflammatory factors.

[0052] Example 2 Inhibitor treatment significantly increased the activity of NK-κB transcription factor; overexpression of SIRT1 reduced the activity of NK-κB transcription factor

[0053] 1. Construction of SIRT1 gene expression vector

[0054] According to the pcDNA3.1-3×Flag C vector information (see Master's thesis, Wang Fei, Molecular mechanism of SPOP regulating innate immunity by negative feedback through degradation of MyD88, DOI: 10.27630 / d.cnki.gznky.2020.000009) and the chicken SIRT1 gene sequence published on NCBI (GenBank: NM_001004767.2), the gene CDS sequence was synthesized by Beijing BGI Biotechnology Co., Ltd. and connected into the vector, and the insertion site was as follows Figure 3 shown.

[0055] 2. Experimental grouping and layout

[0056] The experiment was divided into 5 groups, experimental group (Inhibitor; FLAG-SIRT1, i.e., SIRT1 overexpression plasmid), control group (solvent; empty FLAG), Control group (control vector for dual luciferase experiment), Promoter group (promoter vector), Basic group (Basic vector), and three biological replicates were set in each group. The above vectors and plasmids were purchased from Promega (Beijing) Biotechnology Co., Ltd.).

[0057] According to the experimental design, an appropriate amount of cells of the wild-type HD11 cell line in Example 1 were inoculated into a 24-well plate and cultured in a 37° C., 5% CO 2 incubator.

[0058] 3. Inhibitor treatment / FLAG-SIRT1 transfection of HD11 cell line and LPS treatment

[0059] When the inoculated cell density was about 80%, the gene expression vector SIRT1-pcDNA3.1-3×Flag C was transferred into HD11 cells by liposome transfection. Cell transfection was performed according to the instructions of TransIT-X2® Dynamic Delivery System reagent (Mirus, MIR6000).

[0060] (1) Dilute the required luciferase reporter plasmid with Opti-MEM® medium and mix thoroughly to make a plasmid premix; (2) Add TransIT-X2 reagent to the plasmid premix according to the recommended ratio, mix thoroughly, incubate at room temperature for 15 minutes, and then add the complex to the cell culture dish; (3) SIRT1 Inhibitor (EX527, 10uM) was added to the Inhibitor treatment group; (4) 24 hours after transfection, add LPS (lipopolysaccharide) at a final concentration of 100 ng / ml to the culture dish and treat the cells for 4 hours. After treatment, the luciferase chemiluminescence is detected.

[0061] 4. Dual luciferase reporter gene assay

[0062] The dual-luciferase reporter gene assay was performed according to the instructions of the Dual-Luciferase® Reporter Assay System (Promega, E1910): (1) Cell lysis: Remove the culture medium and add cell lysis buffer to the cell culture plate. After sufficient lysis, ° C, centrifuge at 12000 rpm for 5 min, and aspirate the supernatant for subsequent determination; (2) Prepare Renilla luciferase assay buffer: Calculate the required amount of Renilla luciferase assay buffer based on the volume of 100 µL per sample, and prepare the working solution at a ratio of 1:100 of Renilla luciferase assay substrate to Renilla luciferase assay buffer. (3) Determination of firefly luciferase: Add 100 µL of cell lysate to the ELISA plate, then add 100 µL of firefly luciferase detection reagent, pipette and mix well, and place in an ELISA instrument to detect the chemiluminescence value, which is the firefly luciferase value; (4) Determination of Renilla luciferase: After completing the above steps, add 100 µL of Renilla luciferase detection buffer, mix well according to the above method, and place in a microplate reader to measure the Renilla luciferase value; (5) Calculation: The ratio of the firefly luciferase value to the Renilla luciferase value is the activity of the relevant reporter gene.

[0063] 5. Fluorescence quantitative PCR experiments were used to detect the expression of immune factors after SIRT1 overexpression or inhibitor treatment.

[0064] HD11 cell culture, inhibitor treatment or FLAG-SIRT1 transfection of HD11 cells and LPS treatment were the same as above.

[0065] RNA extraction was carried out according to the method in Example 1.

[0066] 5. Results

[0067] See also Figure 4 , it was shown that after HD11 cells were treated with Inhibitor and induced by LPS, the chemiluminescence intensity showed that the activity of transcription factor NF-κB was significantly increased, indicating that SIRT1 Inhibitor treatment can regulate the expression of downstream immune factors by affecting the activity of transcription factor NF-κB; on the contrary, after expressing FLAG-SIRT1, the chemiluminescence intensity showed that the activity of transcription factor NF-κB was significantly reduced.

[0068] See also Figure 5 , showing the expression of cytokines after fluorescent quantitative PCR detection of Inhibitor and FLAG-SIRT1. After overexpression of SIRT1, IL-1β was significantly reduced; after Inhibitor treatment, IL-1β and IL-8 were significantly upregulated. It can be seen from the figure that SIRT1 can significantly affect the expression of inflammatory factors IL-1β and IL-8.

[0069] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. Application of SIRT1 gene in regulating the innate immune response of birds; in, The poultry is chicken; The reference sequence number of the SIRT1 gene from chicken at NCBI is NM_001004767.2; The avian innate immune response refers to the chicken sensing Gram-negative bacterial infection and initiating an immune response; The applications are for non-disease diagnosis and treatment purposes.

2. The use according to claim 1, characterized in that: The regulation refers to inhibiting the expression of SIRT1 gene at the transcription or translation level to enhance the resistance of chicken mononuclear macrophages to Salmonella.

3. The use according to claim 1, characterized in that: The regulation refers to inhibiting the expression of SIRT1 gene at the transcriptional or translational level to enhance the innate immune response capability of avian animals.

4. Application of SIRT1 gene or protein inhibitors in the preparation of preparations for improving the innate immune response of poultry; in, The poultry is chicken; The reference sequence number of the SIRT1 gene from chicken at NCBI is NM_001004767.2; The avian innate immune response refers to the chicken's perception of Gram-negative bacterial infection and the initiation of an immune response.

5. Use of SIRT1 gene or protein inhibitors in the preparation of preparations for preventing and / or preventing poultry from being infected with Gram-negative bacteria; in, The poultry is chicken; The reference sequence number of the chicken SIRT1 gene on NCBI is NM_001004767.

2.

6. The use according to claim 4 or 5, characterized in that: The inhibitor is selected from a small molecule inhibitor, an oligonucleotide, an antibody, a polypeptide or a fusion protein; Preferably, the inhibitor has the following structure: 。 7. The use according to any one of claims 1 to 5, characterized in that: The Gram-negative bacteria is Salmonella.

8. A drug or composition for preventing and / or treating Gram-negative bacterial infection and improving the innate immune response of poultry, characterized in that: The active ingredient is a SIRT1 gene or protein inhibitor; Wherein, the poultry is chicken; The reference sequence number of the chicken SIRT1 gene on NCBI is NM_001004767.

2.

9. The medicine or composition according to claim 8, characterized in that The inhibitor is selected from a small molecule inhibitor, an oligonucleotide, an antibody, a polypeptide or a fusion protein; Preferably, the inhibitor has the following structure: 。 10. The medicine or composition according to claim 8 or 9, characterized in that: The Gram-negative bacteria is Salmonella.

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