Application of LI0004 protein target in the preparation of drugs to inhibit the proliferation of Lawsoniae-infected cells

Abstract

The present invention discloses the application of the LI0004 protein target in the preparation of a drug for inhibiting the proliferation of Lawsonia infection cells, belonging to the field of biomedicine. IPEC-J2 cells were used to construct an in vitro Lawsonia infection model, and the mechanism of action of the LI0004 protein as a virulence factor in Lawsonia infection cells was studied. The relationship between the LI0004 protein in regulating the activation of the cell cycle signaling pathway and the toxic mechanism was elucidated. The results showed that the LI0004 protein directly bound to the Notch 1 protein, activated the Notch 1 signaling pathway, and then promoted the proliferation of IPEC-J2 cells. The present invention also constructed a mouse model infected with the LI0004 protein. The results showed that the LI0004 protein can produce pathological changes similar to Lawsonia infection in vivo, and the Notch 1 signaling pathway is an important mechanism for the intestinal cell proliferation caused by it.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the use of an inhibitor of the LI0004 protein in preparing a drug targeting the LI0004 protein and inhibiting the proliferation of Lawsonia spp. infected cells. Background Art

[0002] Lawsonia intracellularis (LI), an obligate intracellular parasite, can multiply in large numbers within the intestinal cells of pigs, causing porcine proliferative enteropathy (PPE), characterized by adenomatous hyperplasia of immature intestinal epithelial cells within the crypts of the ileum and colon. Currently, the positive rate of PPE in my country is as high as 79.1%. In recent years, the incidence of digestive tract diseases in pigs caused by Lawsonia infection has increased significantly, resulting in significant economic losses for the pig industry and becoming a major bottleneck restricting its development. However, due to various biological barriers (such as cell membranes), antimicrobial drugs have difficulty entering cells or finding effective targets within cells, thus affecting their therapeutic efficacy. Therefore, studying the virulence factors and pathogenic mechanisms of bacterial infection has become a consensus in the research field of developing new antimicrobial drugs. The Lawsonia LI0004 protein is a Lawsonia hemolysin A-like protein. Multiple studies have shown that hemolysins play a key role in bacterial pathogenesis, particularly in the pathogenesis of bacterial diseases in animals. Hemolysins can disrupt host cell membranes, leading to cell lysis and thus promoting bacterial invasion and spread. Therefore, the presence of hemolysins may have a significant impact on disease severity and progression. However, the toxic mechanism of the Lawsonia spp. hemolysin A-like protein, LhlyA (LI0004), has not been reported. Therefore, it is necessary to investigate the mechanism of action of Lawsonia spp. virulence factors in proliferative enteritis to provide targets for the development of drugs against these virulence factors. Summary of the Invention

[0003] The purpose of the present invention is to provide the use of the LI0004 protein target in the preparation of a drug for inhibiting the proliferation of Lawsoniae-infected cells, so as to solve the problems existing in the above-mentioned prior art. By designing siRNA that inhibits the expression of Notch 1, the binding of Notch 1 protein to LI0004 protein can be inhibited, thereby inhibiting the proliferation effect of LI0004 protein on cells.

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] The present invention provides the use of an inhibitor of LI0004 protein in any of the following:

[0006] (1) Application in the preparation of drugs or reagents for inhibiting cell proliferation;

[0007] (2) Application in the preparation of medicines for treating animal diseases caused by Lawsoniae infection.

[0008] Preferably, the inhibitor of the LI0004 protein includes a product that knocks down or inhibits the expression of Notch1, so as to reduce the binding between the Notch1 protein and the LI0004 protein.

[0009] Preferably, the product for knocking down or inhibiting Notch 1 expression comprises siRNA, wherein the sense strand and antisense strand of the siRNA are shown in any one of the groups of SEQ ID NOs: 1-2, SEQ ID NOs: 3-4, SEQ ID NOs: 5-6 or SEQ ID NOs: 7-8.

[0010] Preferably, the sense strand and antisense strand of the siRNA are as shown in SEQ ID NO: 5-6.

[0011] Preferably, the cells comprise IPEC-J2 cells.

[0012] The present invention also discloses the use of the LI0004 protein target in preparing a drug for inhibiting the proliferation of Lawsoniae-infected cells.

[0013] The present invention also provides use of the LI0004 protein in preparing a cell model or an animal model of cell proliferation.

[0014] Preferably, the cell proliferation model or animal model is constructed by infecting IPEC-J2 cells or animals with LI0004 protein.

[0015] Preferably, the infectious concentration of the LI0004 protein is 0.02-0.08 mg / mL.

[0016] The present invention discloses the following technical effects:

[0017] This study used IPEC-J2 cells and mice as models to construct in vitro and in vivo models, clarifying the mechanism of action of LI0004 protein as a virulence factor in Lawsoniae-infected cells, and elucidating the role of LI0004 protein in regulating the activation of cell cycle signaling pathways and the toxic mechanism during Lawsoniae infection. This provides targets for the development of drugs against Lawsoniae virulence factors, and further provides a scientific basis for the prevention and control of animal diseases caused by Lawsoniae infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. 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 any creative work.

[0019] Figure 1 Western blot results of cell proteins after incubation with LI0004 protein;

[0020] Figure 2 The effect of LI0004 protein on the proliferation of IPEC-J2 cells;

[0021] Figure 3 The effect of LI0004 protein on the expression of Notch pathway proteins; A is a protein graph, B is a protein quantification graph, LI0004 is the purified Lawsonia 0004 protein, and LI is the Lawsonia live vaccine;

[0022] Figure 4 The effect of LI0004 protein on the expression of cell cycle proteins; A is the protein graph, B is the protein quantification graph, LI0004 is the purified Lawsonia 0004 protein, and LI is the Lawsonia live vaccine;

[0023] Figure 5 The effect of LI0004 protein on cell cycle; A is the cell cycle status and the proportion of each phase after flow cytometry detection of the Control group; B is the cell cycle status and the proportion of each phase after flow cytometry detection of the LI0004 protein group;

[0024] Figure 6 To analyze the colocalization of LI0004 and Notch 1 by laser confocal microscopy;

[0025] Figure 7 The expression of Notch 1 after siRNA silencing; upper panel: protein expression; lower panel: protein quantification;

[0026] Figure 8 The effect of Notch 1 silencing on the pathway proteins and cyclin; A is the protein expression graph, and B is the protein quantification graph;

[0027] Figure 9 The results of Co-IP analysis of the interaction between LI0004 protein and Notch 1 molecule; A is SDS-PAGE image, B is Western blot image;

[0028] Figure 10Pathological tissue sections of mouse intestine (400×); C: control group; LI: Lawsonia live vaccine-treated group; LI0004: LI0004 protein-treated group;

[0029] Figure 11 Immunohistochemical detection of Notch 1, ADAM10, c-myc and Cyclin D1 protein expressions in mouse intestinal tissues (400×); C: control group; LI: Lawsonia live vaccine-treated group; LI0004: LI0004 protein-treated group. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] The main experimental materials involved in the following examples are:

[0036] (1) Cells: IPEC-J2 porcine small intestinal epithelial cells, a gift from the National Veterinary Drug Residue Reference Laboratory, Huazhong Agricultural University.

[0037] (2) Main reagents

[0038] CCK-8Cell Counting Kit, catalog number: A311-01, was purchased from Nanjing Novozyme Biotechnology Co., Ltd.;

[0039] ADAM10 Rabbitpab, catalog number: A10438, was purchased from Wuhan Aibote Biotechnology Co., Ltd.;

[0040] Notch 1 Rabbit mAb, catalog number: A19090, was purchased from Wuhan Aibote Biotechnology Co., Ltd.;

[0041] CDK4 Rabbit mAb, catalog number: A23521, was purchased from Wuhan Aibote Biotechnology Co., Ltd.;

[0042] CDK6 Rabbit mAb, catalog number: A0106, was purchased from Wuhan Abotek Biotechnology Co., Ltd.;

[0043] Cyclin D1 RabbitpAb, catalog number: A0310, was purchased from Wuhan Aibote Biotechnology Co., Ltd.;

[0044] Mouse Control IgG, catalog number: AC011, was purchased from Wuhan Aibote Biotechnology Co., Ltd.;

[0045] lipo8000 TM Transfection reagent, catalog number: C0533, was purchased from Beyotime Biotechnology Co., Ltd.;

[0046] LI0004 Rabbit mAb, homemade in the laboratory (preparation method see "CN117659184A A polyclonal antibody to the intracellular Lawsonia LI0004 protein, its preparation method and application");

[0047] HRP GoatAnti-Rabbit IgG (H+L), catalog number AS014, was purchased from Wuhan Aibote Biotechnology Co., Ltd.;

[0048] 488-conjugated Goat anti-Mouse IgG (H+L), catalog number: AS037, purchased from Wuhan Sanying Technology Co., Ltd.;

[0049] Cy3-conjugated Goat anti-Rabbit IgG (H+L), catalog number: AS007, purchased from Wuhan Sanying Technology Co., Ltd.;

[0050] Protein A / G Magnetic Beads, catalog number: HY-K0202, purchased from Msd Chem Express;

[0051] Opti-MEM, catalog number: 31985-070, purchased from Thermo Fisher Scientific Inc.;

[0052] BSAalbumin fraction V, product number: 4240GR100, was purchased from Saiguo Biotechnology Co., Ltd.;

[0053] 10% SDS-PAGE denaturing acrylamide colored gel rapid preparation kit, catalog number: C601102, purchased from Sangon Biotech (Shanghai) Co., Ltd.;

[0054] Bacterial DNA extraction kit, catalog number: DP201101X, was purchased from Tiangen Biotechnology Co., Ltd.;

[0055] DH5α chemically competent cells, catalog number: C502-02, were purchased from Nanjing Novozyme Biotechnology Co., Ltd.

[0056] Rosetta (DE3) Chemically Competent Cell, catalog number: EC1010S, purchased from Shanghai Weidi Biotechnology Co., Ltd.;

[0057] Porcine ileitis live vaccine Ileitis, catalog number: 6040290B, was purchased from Boehringer Ingelheim Animal Health GmbH;

[0058] Endotoxin-free plasmid miniprep kit (catalog number: DP118) was purchased from Tiangen Biotechnology Co., Ltd.

[0059] Example 1

[0060] 1. Recovery and culture of IPEC-J2 cells

[0061] Remove the frozen IPEC-J2 cells from the liquid nitrogen tank and quickly place them in a 37°C water bath, shaking the cryovial. Once the cell suspension has thawed, transfer the cells to a centrifuge tube. Centrifuge at 1000 rpm for 4 minutes, discard the supernatant, and invert the tube for 1 minute to remove as much of the suspension as possible. Resuspend the cells in complete medium and transfer them to a cell culture flask. Incubate at 37°C with 5% CO2. Use in subsequent experiments only when the cells are growing well and at an appropriate density.

[0062] 2. Passaging and cryopreservation of IPEC-J2 cells

[0063] Passaging: When cell confluence reaches 90% or greater, discard the complete medium and wash the cells twice with 1 mL of PBS. Digest the cells with trypsin for 30 seconds, discard the trypsin, and place the cell flask in a CO2 incubator for 4 minutes. Terminate the digestion by adding complete medium containing serum. Divide the cells equally between two cell culture flasks, make up to 5 mL with complete medium, and continue culturing in a CO2 incubator.

[0064] Cryopreservation: After digesting the cells with trypsin, add complete culture medium to terminate digestion and transfer the cell suspension to a centrifuge tube. Centrifuge at 1000 rpm for 4 minutes, discard the supernatant, resuspend the cells in 1 mL of freezing buffer, transfer to a cryopreservation tube, label the cell name, generation number, and freezing date, place in a -80°C freezer overnight, and then transfer to a liquid nitrogen tank.

[0065] 3. Detection of cell viability by CCK8 assay

[0066] (1) When the cell density reaches above 80%, add trypsin for 30 seconds, dry-dissolve for 3 minutes, and add 2 mL of complete culture medium to mix thoroughly.

[0067] (2) Add 5000 cells to each well of a 96-well plate and incubate in a CO2 incubator for 12 h.

[0068] (3) Add 100 μL of culture medium containing the corresponding concentration of LI0004 protein to each well and incubate in a CO2 incubator for 24 h.

[0069] (4) Take 1.1 mL of CCK8 solution and add 11 mL of DMEM basal medium, mix well, add the mixed CCK8 solution to each well, and place in a CO2 incubator for incubation for 2 h.

[0070] (5) Use an enzyme-labeled instrument to measure the absorbance at 450 nm.

[0071] 4. Western blot detection of protein expression

[0072] The treated IPEC-J2 cells were washed twice with 2 mL of PBS and cell lysis buffer was added. The IPEC-J2 cells in the six-well plate were washed twice with pre-cooled PBS. 200 μL of cell lysis buffer was added to each well and the cells were lysed on ice for 20 minutes. The adherent cells were scraped off with a sterilized cell scraper, transferred to a centrifuge tube, and placed on ice for ultrasonic disruption of the cells. The ultrasonic conditions were: 40% power, a total ultrasonic time of 30 seconds, 10 seconds each time, and an interval of 15 seconds between two times. The protein concentration was then determined using a kit.

[0073] Western blot identification of LI0004 protein:

[0074] (1) After SDS-PAGE electrophoresis, prepare filter paper and PVDF membrane.

[0075] (2) Place the PVDF membrane in methanol for 15 minutes, then soak it in electrotransfer buffer for 20 minutes. Soak the filter paper in a small amount of electrotransfer buffer in a dish for 30 minutes.

[0076] (3) Install the transfer device, use a glass rod to remove bubbles, and align the PVDF membrane on the filter paper to ensure there are no bubbles. Cut off the stacking gel of the PAGE gel and cut the separation gel into appropriate sizes. Rinse with ultrapure water and soak in transfer buffer for 20 minutes. Place the gel accurately on the PVDF membrane and remove bubbles.

[0077] (4) Place another piece of filter paper on top of the glue, with the graphite side of the upper electrode facing down and covering the sandwich. Adjust the current and voltage according to the size of the glue area.

[0078] (5) Blocking: Rinse the membrane in TBST three times, 5 min each time. Then, incubate the membrane in blocking solution at room temperature for 3 h.

[0079] (6) Dilute the primary antibody at 1:5000 in TBST and incubate the PVDF membrane in the primary antibody dilution solution at 4°C overnight.

[0080] (7) Remove the PVDF membrane and wash it three times with TBST (15 min each time). Add the secondary antibody diluted in blocking solution (1:5000 dilution) and incubate at room temperature for 1 hour. Wash it three times with TBST (15 min each time).

[0081] (8) Remove the PVDF membrane, use filter paper to absorb excess water on the membrane, and soak the membrane in the color developing solution for 2 minutes to develop the color.

[0082] 5. Flow cytometry detection of cell cycle

[0083] (1) IPEC-J2 cells were treated with LI0004 (prepared using the method described in the invention patent "CN117659184A: A polyclonal antibody against the intracellular Lawsonia LI0004 protein, its preparation method, and its application") at a final concentration of 0.02 mg / mL for 24 h. The cells were then trypsinized, collected into a centrifuge tube, and centrifuged at 1000 g for 5 min. The supernatant was discarded. The cells were resuspended in pre-chilled PBS and centrifuged again, and the supernatant was discarded.

[0084] (2) Treat the cells with pre-chilled 70% ethanol for 12 h. Centrifuge at 1000 rpm for 5 min and discard the supernatant. Add pre-chilled PBS and resuspend the cells. Centrifuge again and discard the supernatant.

[0085] (3) Add 0.5 mL of propidium iodide staining solution to the cell sample, mix gently, and incubate at 37°C in the dark for 30 min.

[0086] (4) Red fluorescence was detected using a flow cytometer at an excitation wavelength of 488 nm.

[0087] 6. Indirect immunofluorescence double labeling test

[0088] (1) Place a round coverslip soaked in anhydrous ethanol in a 12-well plate, wash three times with PBS, and seed 30,000 to 50,000 cells per well. After the cells adhere, add 0.02, 0.04, and 0.08 mg / mL of LI0004 protein and incubate for 24 hours.

[0089] (2) Aspirate the complete culture medium, wash three times with PBS, add paraformaldehyde fixative, and fix in a 4°C refrigerator for 20 min.

[0090] (3) Add 0.2% TritonX-100 and incubate at room temperature for 10 min, then wash three times with PBS.

[0091] (4) 5% BSA, incubate at 37°C for 30 min, remove the blocking solution, and directly add the primary antibody mixture (Notch 1 Rabbit mAb and His-tag Mouse mAb) at a dilution ratio of 1:200, and incubate at 4°C overnight.

[0092] (5) Recover the primary antibody mixture and wash with PBST three times. Add the secondary antibody ( The cells were incubated with a mixture of 488-conjugated Goat anti-Mouse IgG and Cy3-conjugated Goat anti-Rabbit IgG at 37°C in the dark for 1 h and then washed three times with PBST.

[0093] (6) Remove the secondary antibody mixture, wash three times with PBST, add DAPI working solution, incubate at room temperature for 5 min, wash three times with PBST, and observe and collect images under a fluorescence microscope.

[0094] 7. Co-immunoprecipitation assay

[0095] (1) Wash cells twice with pre-cooled PBS.

[0096] (2) Add 10 mL of pre-cooled PBS to scrape the cells, centrifuge at 1500 rpm for 5 minutes, and collect the cells.

[0097] (3) Add 1 mL of RIPA (weak) lysis buffer to resuspend the cells and lyse on ice for 15 min.

[0098] (4) Centrifuge at 3000 rpm for 15 min, transfer the supernatant to a new EP tube, take 100 μL as the control group (input group), and divide the rest into two tubes, each containing 400 μL.

[0099] (5) Add His-tag mAb and IgG (Mouse Control IgG) antibodies to two tubes of cell lysate (His group and IgG group) respectively and incubate on a shaker at 4°C overnight.

[0100] (6) Pipette 50 μL of magnetic beads and place on a magnetic rack. Discard the supernatant, wash twice with pre-chilled PBS, and resuspend in 100 μL of PBS. Add 50 μL of magnetic beads to each cell lysate. Incubate on a shaker at 4°C for 3 h.

[0101] (7) Place the two centrifuge tubes on a magnetic rack, discard the supernatant, wash three times with 500 μL of pre-cooled PBS, and finally remove the PBS.

[0102] (8) Add 50 μL of 1× protein loading buffer (40 μL of PBS + 10 μL of 5× protein loading buffer) and denature at 98°C for 5 min.

[0103] The centrifuge tube was placed on a magnetic rack, and the supernatant was collected and subjected to SDS-PAGE and Western blot assays (Notch1 Rabbit mAb (Cat. No. A19090, purchased from Wuhan Aibote Biotechnology Co., Ltd.) and LI0004 Rabbit mAb).

[0104] 8. Notch 1-siRNA interference assay

[0105] Notch 1-siRNA was synthesized by Genema, and the sequences of the four Notch 1-siRNAs are shown in Table 1. The four siRNAs were transfected into IPEC-J2 cells, and the cell proteins were collected. The expression of Notch 1 was detected by Western blot.

[0106] Table 1 siRNA sequences

[0107]

[0108] IPEC-J2 cells were divided into a control group, a Notch 1-siRNA group, a control group + LI0004 protein group, and a Notch 1-siRNA group + LI0004 protein group. Transfection was performed at a ratio of siRNA:lipo8000 = 7.5 μL:5 μL. Opti-MEM was added first, followed by the corresponding amount of siRNA, and then lipo8000 transfection reagent was added. Mix gently, let stand for 20 minutes, and then evenly distribute the reagents into six-well plates. Cultures were continued for 5 hours, then replaced with 10% complete medium without anti-antibody. Cultures were continued for 19 hours. LI0004 protein was added for 24 hours, and protein samples were collected. Protein was quantified by BCA assay and analyzed by Western blot.

[0109] 9. Data Analysis

[0110] Graphpad Prism 7.0 statistical software was used for plotting, and SPSS 18.0 was used for data processing. One-way ANOVA was used to test for significance between multiple groups, and Student's t-test was used to test for significance between two groups. For the comparison between the Notch 1-siRNA + LI0004 protein group and the NC + LI0004 protein group, # = p < 0.05 indicates a significant difference, and ## = p < 0.01 indicates an extremely significant difference. For the comparison between the NC + LI0004 protein and Notch 1-siRNA groups and the NC group, * = p < 0.05 indicates a significant difference, and ** = p < 0.01 indicates an extremely significant difference.

[0111] 10. Test results

[0112] 10.1 Effect of LI0004 protein on IPEC-J2 cell activity

[0113] After the cells were treated with LI0004 protein for 24 h, the LI0004 protein was detected by Western blot. Figure 1 As shown in Figure 2, LI0004 protein can be detected in cell proteins, and the trend of protein results is consistent with the concentration trend of LI0004 protein, proving that LI0004 protein can enter IPEC-J2 cells and play a role. Figure 2 As shown in Figure 3, 0.02, 0.04, and 0.08 mg / mL of LI0004 protein can promote the proliferation of IPEC-J2 cells in a dose-dependent manner (p < 0.05). Therefore, subsequent experiments used 0.02, 0.04, and 0.08 mg / mL of LI0004 protein to incubate IPEC-J2 cells.

[0114] 10.2 Effect of LI0004 protein on the expression of Notch pathway-related proteins

[0115] In order to study the effect of LI0004 protein on the proliferation of IPEC-J2 cells, the present invention used Lawsonia live vaccine and different concentrations of LI0004 protein to treat IPEC-J2 cells, and used Western blot to detect the effect of LI0004 protein on Notch pathway related proteins. The results are as follows: Figure 3 The results showed that compared with the blank group, treatment of IPEC-J2 cells with Lawsonia live vaccine and 0.02, 0.04, and 0.08 mg / mL of LI0004 protein significantly upregulated the expression of Notch pathway-related proteins (p < 0.01), indicating that Lawsonia and LI0004 protein can activate the expression of Notch 1, ADAM10, and Hes-1 proteins in the Notch pathway, thereby affecting cell proliferation.

[0116] 10.3 Effects of LI0004 Protein on Cell Cycle

[0117] In order to study the effect of LI0004 protein on cell cycle, the present invention used Lawsonia live vaccine IPEC-J2 cells to be inoculated into six-well plates, treated IPEC-J2 cells with different concentrations of LI0004 protein, and used Western blot to detect the effect of LI0004 protein on cell cycle-related proteins. The results are as follows Figure 4 As shown, the results showed that compared with the blank group, after IPEC-J2 cells were treated with Lawsonia live vaccine and 0.02, 0.04 and 0.08 mg / mL LI0004 protein, the expression of cell cycle-related proteins Cyclin D1, CDK4, CDK6, and c-myc were significantly upregulated (p < 0.01).

[0118] IPEC-J2 cells were seeded into culture dishes and treated with LI0004 protein for 24 hours before being collected for flow cytometry analysis. Figure 5 As shown, compared with the control group, the proportion of S phase cells in the LI0004-treated group was significantly increased, indicating that LI0004 protein treatment promoted the proliferation of IPEC-J2 cells.

[0119] 10.4 Indirect immunofluorescence double labeling assay to analyze the colocalization of LI0004 protein and Notch 1

[0120] LI0004 protein was treated with IPEC-J2 cells, and the distribution of LI0004 protein in IPEC-J2 cells, Notch 1 expression and co-localization analysis were detected by indirect immunofluorescence double labeling assay. Figure 6As shown, the green fluorescence emitted by the LI0004 protein colocalizes closely with the red fluorescence of Notch 1, indicating that the LI0004 protein is primarily distributed in the nucleus and cytoplasm. Following LI0004 treatment, Notch 1 expression levels increased significantly with increasing LI0004 concentrations, and Notch 1 gradually translocated to the nucleus. This suggests that LI0004 directly binds to Notch 1 after infection of IPEC-J2 cells, increasing Notch 1 expression and promoting its translocation to the nucleus.

[0121] 10.5 Notch 1 Interference Test

[0122] Four siRNAs (Notch 1-siRNA 2513, 1928, 2291, 3591) were respectively applied to IPEC-J2 cells, cell proteins were collected, and the expression of Notch 1 was detected by Western blot. Figure 7 As shown, Notch 1-siRNAs of different sequences can partially interfere with the expression of Notch 1, among which Notch 1-siRNA-2291 has the best effect in interfering with Notch 1 expression (p < 0.05). Therefore, Notch 1-siRNA-2291 was selected to act on IPEC-J2 cells to interfere with Notch 1 expression for subsequent experiments.

[0123] The Notch 1 interference assay was used to investigate the mechanism by which LI0004 protein induced IPEC-J2 cell proliferation. After Notch1-siRNA transfection, Western blot assay was used to detect the effects of LI0004 protein on Notch pathway and cell cycle-related proteins. Figure 8 As shown in the results, interference with Notch 1 expression significantly reduced the expression of Notch 1, Hes-1, Cyclin D3, and c-myc (p < 0.01), while LI0004 protein significantly upregulated the expression of Notch 1 and cell cycle-related proteins (p < 0.01). These results indicate that Notch 1 knockdown inhibited the effect of LI0004 protein on cell proliferation.

[0124] 10.6 Co-IP analysis of the interaction between LI0004 protein and Notch 1 molecule

[0125] To further investigate the interaction between LI0004 protein and Notch 1, the present invention treated IPEC-J2 cells with purified His-tagged LI0004 protein. After 24 hours of treatment, the cell proteins were collected and the interaction between LI0004 protein and Notch 1 was confirmed by Co-IP. The complex of LI0004 protein and its interacting proteins was obtained by magnetic bead adsorption and Western blot was used to detect whether Notch 1 was present. Figure 9 As shown, Notch 1 antibody detection revealed a Notch 1 band in the precipitated complex, while the IgG control precipitated complex only had a weak, non-specific band. These results indicate that LI0004 protein directly binds to Notch 1 protein, activates the Notch 1 signaling pathway, and thereby promotes IPEC-J2 cell proliferation.

[0126] From the above results, it can be seen that LI0004 protein can cause increased expression of Notch pathway and cyclin in IPEC-J2 cells in vitro, proving that LI0004 protein can serve as a key virulence factor of Lawsoniae to infect IPEC-J2 cells.

[0127] Example 2 Effect of LI0004 protein on host cell proliferation in vivo

[0128] 1. Experimental Animals

[0129] BALB / c female mice, 4 weeks old, SPF grade, were purchased from Hubei Provincial Laboratory Animal Research Center.

[0130] 2. Test methods

[0131] 2.1 Trial Grouping

[0132] Thirty 4-week-old SPF female BALB / c mice were randomly divided into five groups: control group, Lawsonia live vaccine (hereinafter referred to as LI) group, LI0004 protein treatment group, LI+valnemulin group, and LI0004+valnemulin group, with 6 mice in each group. Each mouse in the LI group was injected with 250 μL of 10 5 The LI0004 group received a live Lawsonia suis vaccine (TCID50 / mL) at a protein dose of 250 μg per mouse. Valnemulin was administered orally at a dose of 3 mg / kg bw per mouse. The experimental period was 14 days. After 14 days, mice were sacrificed, and ileal and cecal tissues were collected. The intestinal tissue samples from each segment were divided into three aliquots. One aliquot was fixed in 10% neutral formalin solution for histopathological section preparation, and the other two aliquots were stored at -80°C for PCR analysis.

[0133] 2.2 Pathological tissue testing

[0134] (1) Place mouse ileum tissue in paraformaldehyde fixative and fix for 24 hours.

[0135] (2) PBS was used to remove paraformaldehyde, and the ileum tissue was dehydrated using a dehydrator.

[0136] (3) After soaking in xylene for 40 minutes, the tissue was immersed in liquid paraffin for a long time. The mouse intestinal tissue was embedded in an embedding machine and then sectioned.

[0137] (4) After the cut serial sections are fully unfolded in a water bath, the moisture on the slides is removed and the slides are baked.

[0138] (5) Dewax the sections in xylene solution, 100%, 95%, 85%, and 70% ethanol to water.

[0139] (6) Soak the slides of intestinal tissue in hematoxylin and eosin staining solutions in turn. After confirming that the staining is successful, rinse with distilled water for 1 minute.

[0140] (7) Remove excess water from the intestinal tissue sections, dehydrate them in a dehydrator, and then seal the sections.

[0141] 2.3 Immunohistochemistry

[0142] (1) Dewaxing of tissue sections: Cut the wax block into 4 μm slices, soak in xylene for 5 min, soak in anhydrous ethanol for 5 min, then soak in 95%, 90%, 80%, and 70% ethanol for 2 min, and rinse with PBS three times, 5 min each time.

[0143] (2) Prepare 3% hydrogen peroxide solution and place the slices in the solution at room temperature in the dark for 10 minutes.

[0144] (3) Use a microwave to boil the citric acid buffer, add the slices and continue heating for 2 minutes, and repeat twice (the interval between the two times is 8 minutes).

[0145] (4) Prepare 3% BSA solution and incubate the slices in the solution at room temperature for 30 minutes.

[0146] (5) Add the primary antibody dilution solution to the slices and incubate in a 37°C incubator for 30 min (place the slices in a humidified box to avoid antibody loss).

[0147] (6) Wash the sections three times with PBS, add rabbit IgG drops onto the sections, and incubate in a 37°C incubator for 30 min (the sections are placed in a wet box).

[0148] (7) Incubate the treated sections in the colorimetric solution at room temperature, monitoring the incubation time under a microscope. Wash the sections three times with distilled water, each for 1 min.

[0149] (8) Lightly counterstain with thiazolin for 10 seconds and rinse with distilled water for 10 minutes for blueing.

[0150] (9) Dehydrate, transparentize, and seal the slides.

[0151] 3. Experimental results and analysis

[0152] 3.1 Mouse intestinal pathological tissue sections

[0153] After the mice were killed, the ileum tissue was collected and fixed to prepare intestinal pathological tissue sections. Figure 10 As shown, compared with the blank control group, the group infected with Lawsonia live vaccine (LI) showed minimal crypt cell proliferation. However, mice infected with LI0004 protein experienced shortened intestinal villi and significantly thickened intestinal walls. Treatment with valnemulin alleviated crypt cell proliferation and villus damage, demonstrating that LI0004 protein can promote intestinal cell proliferation in vivo.

[0154] 3.2 Effects of Lawsonia and LI0004 protein on protein expression in mouse intestinal tissue

[0155] In the above experiments, LI0004 protein can promote the expression of Notch 1 pathway proteins and cell cycle proteins in vitro. Therefore, in order to explore the effects of Lawsoniae and LI0004 protein on the expression of related proteins in mouse intestinal tissue, immunohistochemical staining was used to determine the levels of Notch 1, ADAM10, c-myc and Cyclin D1 in mouse intestinal tissue. Figure 11 As shown, the results showed that the positive reactions of Notch 1, ADAM10, c-myc and Cyclin D1 increased after the mouse intestine was infected with LI0004 protein and Lawsonia live vaccine.

[0156] From the above experimental results, it can be seen that LI0004 protein can produce pathological changes similar to Lawsonia infection in the body, and the Notch 1 signaling pathway is an important mechanism for the intestinal cell proliferation caused by it.

[0157] In summary, Notch 1 may be a key target of LI0004 in intestinal epithelial cell proliferation. This study, at the molecular level, clarifies the relationship between LI0004 and Notch 1 in regulating Notch signaling pathway activation and host cell cycle regulation. This study also provides a preliminary understanding of the mechanism of action of LI0004 in Lawsoniae-induced host cell proliferation and cell cycle toxicity, laying the foundation for the development of anti-Lawsoniae drugs targeting LI0004.

[0158] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Use of an inhibitor of LI0004 protein in any of the following: (1) Use in the preparation of drugs or reagents for inhibiting the proliferation of Lawsonia infected cells; (2) Use in the preparation of medicines for treating animal diseases caused by Lawsoniae infection; The inhibitor of the LI0004 protein is siRNA, and the sense strand and antisense strand of the siRNA are shown in SEQ ID NO: 5-6.

Images