Application of LI0004 protein target spot in preparation of medicine for inhibiting Lawsonia infected cell proliferation

Abstract

The invention discloses application of an LI0004 protein target spot in preparation of a medicine for inhibiting Lawsonia infection cell proliferation, and belongs to the field of biological medicine. IPEC-J2 cells are adopted, an in-vitro Lawsonia infection model is constructed, the action mechanism of LI0004 protein as a virulence factor in Lawsonia infected cells is researched, the relationship of the LI0004 protein in regulating cell cycle signal channel activation and the virulence action mechanism is clarified, and the result shows that the LI0004 protein is directly combined with Notch 1 protein, the Notch 1 signal channel is activated, and then IPEC-J2 cell proliferation is promoted. The invention also constructs an LI0004 protein infected mouse model, the result shows that the LI0004 protein can generate pathological change similar to Lawsonia infection in vivo, and the Notch 1 signal channel is an important action mechanism of intestinal cell proliferation caused by the Notch 1 signal channel.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the use of an inhibitor of LI0004 protein in preparing a drug for inhibiting the proliferation of Lawsonia infection cells by using a LI0004 protein target. Background Art

[0002] Lawsonia intracellularis (LI), as an obligate intracellular parasite, can multiply in large numbers in pig intestinal cells, causing a porcine proliferative enteropathy (PPE) characterized by adenomatous hyperplasia of immature intestinal epithelial cells in the crypts of the ileum and colon. At present, the positive rate of this disease in my country is as high as 79.1%. In recent years, the digestive tract diseases of pigs caused by Lawsonia infection have increased significantly, causing huge economic losses to the pig industry and becoming an important bottleneck restricting the development of the pig industry. However, due to the effects of various biological barriers in the body (such as cell membranes, etc.), it is difficult for antibacterial drugs to enter the cell, or they cannot find effective targets in the cell, thus affecting the therapeutic effect of the drug. Therefore, studying the virulence factors and pathogenic mechanisms of bacterial infection has become a consensus in the research field of developing new antibacterial drugs. Lawsonia LI0004 protein is a Lawsonia hemolysin A-like protein. Many studies have shown that hemolysin plays an important role in bacterial pathogenesis, especially in the pathogenesis of bacterial diseases in animals. Hemolysins can destroy the cell membrane of host cells, causing cell lysis, thereby promoting bacterial invasion and spread. Therefore, the presence of hemolysins may have an important impact on the severity and progression of the disease. However, the toxic mechanism of the Lawsonia solani hemolysin A-like protein LhlyA (LI0004) has not been reported. Therefore, it is necessary to study the mechanism of proliferative enteritis caused by Lawsonia solani virulence factors to provide targets for the development of drugs against Lawsonia solani virulence factors. Summary of the invention

[0003] The purpose of the present invention is to provide an application of the LI0004 protein target in the preparation of a drug for inhibiting the proliferation of Lawsonia 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) Use in the preparation of drugs or reagents for inhibiting cell proliferation;

[0007] (2) Use 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 to reduce the binding of the Notch1 protein to 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 invention also discloses the application of the LI0004 protein target in preparing a drug for inhibiting the proliferation of Lawsonia infection cells.

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

[0014] Preferably, the cell proliferation cell 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] The present invention constructs in vivo and in vitro models using IPEC-J2 cells and mice as models, clarifies the mechanism of action of LI0004 protein as a virulence factor in Lawsonia infected cells, and clarifies the role of LI0004 protein in regulating the activation of cell cycle signaling pathways and the toxic action mechanism during Lawsonia infection, providing targets for the development of drugs against Lawsonia virulence factors, and further providing a scientific basis for the prevention and control of animal diseases infected with Lawsonia. 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 drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[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 of the Control group after flow cytometry detection and the proportion of each phase; B is the cell cycle status of the LI0004 protein group after flow cytometry detection and the proportion of each phase;

[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 figure: protein expression diagram; lower figure: protein quantification diagram;

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

[0027] Fig. 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] Fig.10Mouse intestinal pathological tissue sections (400×); C: control group; LI: Lawsonia live vaccine treatment group; LI0004: LI0004 protein treatment group;

[0029] Fig.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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content 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 of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary 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, donated by the National Veterinary Drug Residue Reference Laboratory of Huazhong Agricultural University.

[0037] (2) Main reagents

[0038] CCK-8Cell Ccounting Kit, catalog number: A311-01, 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, purchased from Wuhan Abotec Biotechnology Co., Ltd.;

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

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

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

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

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

[0046] LI0004 Rabbit mAb, homemade in the laboratory (the preparation method is described in “CN117659184A A polyclonal antibody against the intracellular Lawsonia LI0004 protein and its preparation method and application”);

[0048] HRP GoatAnti-Rabbit IgG (H+L), catalog number AS014, was purchased from Wuhan Abotek Biotechnology Co., Ltd.;

[0049] 488-conjugated Goat anti-Mouse IgG (H+L), catalog number: AS037, purchased from Wuhan Sanying Technology Co., Ltd.;

[0050] Cy3-conjugated Goat anti-Rabbit IgG (H+L), catalog number: AS007, purchased from Wuhan Sanying Technology Co., Ltd.;

[0051] Protein A / G Magnetic Beads, catalog number: HY-K0202, purchased from Msd Chem Express;

[0052] Opti-MEM, catalog number: 31985-070, purchased from Thermo Fisher Scientific Inc.;

[0053] BSAalbumin fraction V, catalog number: 4240GR100, purchased from Saiguo Biotechnology Co., Ltd.;

[0054] 10% SDS-PAGE denaturing acrylamide colored gel rapid preparation kit, catalog number: C601102, purchased from Sangon Biotechnology (Shanghai) Co., Ltd.;

[0055] Bacterial DNA extraction kit, catalog number: DP201101X, purchased from Tiangen Biotechnology Co., Ltd.;

[0056] DH5α chemically competent cells, catalog number: C502-02, purchased from Nanjing Novozyme Biotechnology Co., Ltd.;

[0057] Rosetta (DE3) Chemically Competent Cell, catalog number: EC1010S, purchased from Shanghai Weidi Biotechnology Co., Ltd.;

[0058] Swine Ileitis Live Vaccine Ileitis, catalog number: 6040290B, was purchased from Boehringer Ingelheim Animal Health;

[0059] Endotoxin-free plasmid miniprep kit (catalog number: DP118) was purchased from Tiangen Biotechnology Co., Ltd.

[0060] Example 1

[0061] 1. Recovery and culture of IPEC-J2 cells

[0062] Take out the frozen IPEC-J2 cells from the liquid nitrogen tank, quickly place them in a water bath at 37°C, and shake the cryotubes constantly. After the cell cryopreservation solution is thawed, transfer them to a centrifuge tube, centrifuge at 1000rpm for 4min, discard the supernatant, invert the centrifuge tube for 1min, and remove as much cryopreservation solution as possible. Add complete culture medium to the centrifuge tube to resuspend the cells, transfer them to a cell culture bottle, and culture them at 37°C and 5% CO2. When the cells are in good growth condition and the density is appropriate, they can be used for subsequent experiments.

[0063] 2. Passaging and cryopreservation of IPEC-J2 cells

[0064] Passaging: When the cell confluence reaches more than 90%, discard the complete medium, wash the cells twice with 1 mL PBS, digest the cells with trypsin for 30 seconds, pour out the trypsin, and place the cell bottle in a CO2 incubator for 4 minutes. Add complete medium containing serum to terminate digestion, divide it equally into two cell culture bottles, add complete medium to 5 mL, and continue culturing in a CO2 incubator.

[0065] Cryopreservation: After digesting the cells with trypsin, add complete culture medium to terminate digestion, transfer the cell suspension to a centrifuge tube, centrifuge at 1000rpm for 4min, discard the supernatant, resuspend the cells in 1mL of freezing solution, transfer to a cryopreservation tube, mark the cell name, generation number and freezing date, place in a -80℃ refrigerator overnight, and then transfer to a liquid nitrogen tank.

[0066] 3. CCK8 assay to detect cell viability

[0067] (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 by pipetting.

[0068] (2) Add 5000 cells to each well of a 96-well plate and incubate in a CO2 incubator for 12 h.

[0069] (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.

[0070] (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.

[0071] (5) Use an ELISA reader to measure the absorbance at 450 nm.

[0072] 4. Western blot detection of protein expression

[0073] The treated IPEC-J2 cells were washed twice with 2 mL PBS and cell lysis solution was added; the IPEC-J2 cells in the six-well plate were washed twice with pre-cooled PBS; 200 μL of cell lysis solution was added to each well and lysed on ice for 20 min; 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 s, 10 s each time, and an interval of 15 s between two times, and then the protein concentration was determined using a kit.

[0074] Western blot identification of LI0004 protein:

[0075] (1) After SDS-PAGE electrophoresis, prepare filter paper and PVDF membrane.

[0076] (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 for 30 minutes.

[0077] (3) Install the transfer device, use a glass rod to remove bubbles, and align the PVDF membrane on the filter paper to ensure that 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.

[0078] (4) Place another piece of filter paper on top of the glue and align it with the graphite side of the upper electrode facing down on the sandwich. Adjust the current and voltage according to the size of the glue area.

[0079] (5) Blocking: The membrane was rinsed in TBST three times, 5 min each time, and then incubated in blocking solution at room temperature for 3 h.

[0080] (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.

[0081] (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.

[0082] (8) Take out 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.

[0083] 5. Flow cytometry detection of cell cycle

[0084] (1) After treating IPEC-J2 cells with LI0004 (prepared by the method described in the invention patent "CN117659184A: a polyclonal antibody against LI0004 protein of Lawsonia intracellularis and its preparation method and application") at a final concentration of 0.02 mg / mL for 24 h, the cells were digested with trypsin, collected into a centrifuge tube, centrifuged at 1000 g for 5 min, and the supernatant was discarded. After adding pre-cooled PBS to resuspend the cells, the cells were centrifuged again and the supernatant was discarded.

[0085] (2) Treat the cells with pre-cooled 70% ethanol for 12 h. Centrifuge at 1000 rpm for 5 min and discard the supernatant. Add pre-cooled PBS and resuspend the cells. Centrifuge again and discard the supernatant.

[0086] (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.

[0087] (4) Red fluorescence was detected using a flow cytometer at an excitation wavelength of 488 nm.

[0088] 6. Indirect immunofluorescence double labeling test

[0089] (1) Place a round cover glass soaked in anhydrous ethanol in a 12-well plate, wash it 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 culture for 24 hours.

[0090] (2) Aspirate the complete culture medium, wash three times with PBS, add paraformaldehyde fixative, and fix in a 4°C refrigerator for 20 min.

[0091] (3) Add 0.2% TritonX-100 and incubate at room temperature for 10 min, then wash three times with PBS.

[0092] (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.

[0093] (5) Recover the primary antibody mixture and wash with PBST three times. Add the secondary antibody ( The cells were mixed with 488-conjugated Goat anti-Mouse IgG and Cy3-conjugated Goat anti-Rabbit IgG), incubated at 37°C in the dark for 1 h, and washed three times with PBST.

[0094] (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.

[0095] 7. Co-immunoprecipitation assay

[0096] (1) Wash cells twice with pre-cooled PBS.

[0097] (2) Add 10 mL of pre-cooled PBS to scrape the cells and centrifuge at 1500 rpm for 5 min to collect the cells.

[0098] (3) Add 1 mL of RIPA (weak) lysis buffer to resuspend the cells and lyse on ice for 15 min.

[0099] (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, 400 μL each.

[0100] (5) Add His antibody (His-tag mAb) and IgG (Mouse Control IgG) antibody to two tubes of cell lysate (His group and IgG group), respectively, and incubate on a shaker at 4°C overnight.

[0101] (6) Pipette 50 μL of magnetic beads, place on a magnetic rack, discard the supernatant, wash twice with pre-cooled PBS, and resuspend with 100 μL PBS. Add 50 μL of magnetic beads to each group of cell lysate. Incubate on a shaker at 4°C for 3 h.

[0102] (7) Place two centrifuge tubes on a magnetic rack, discard the supernatant, wash three times with 500 μL pre-cooled PBS, and finally remove the PBS.

[0103] (8) Add 50 μL 1× protein loading buffer (40 μL PBS + 10 μL 5× protein loading buffer) and denature at 98°C for 5 min.

[0104] The centrifuge tube was placed on a magnetic rack, and the supernatant was collected and subjected to SDS-PAGE test and Western blot test (Notch1 Rabbit mAb (Cat. No.: A19090, purchased from Wuhan Abotek Biotechnology Co., Ltd.) and LI0004 Rabbit mAb).

[0105] 8. Notch 1-siRNA interference assay

[0106] Notch 1-siRNA was synthesized by Jima Gene, 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 to detect the expression of Notch 1 by Western blot assay.

[0107] Table 1 siRNA sequences

[0108]

[0109] IPEC-J2 cells were divided into control group, Notch 1-siRNA group, control group + LI0004 protein group, Notch 1-siRNA group + LI0004 protein group. Transfection was performed according to siRNA:lipo8000=7.5μL:5μL, Opti-MEM was added first, then the corresponding amount of siRNA was added, and then lipo8000 transfection reagent was added, gently mixed, and the above reagents were evenly distributed into six-well plates after standing for 20 minutes, and then cultured for 5 hours and replaced with 10% complete medium without double antibody, and cultured for 19 hours. After adding LI0004 protein for 24 hours, protein samples were collected, and Western blot detection was performed after BCA protein quantification.

[0110] 9. Data Analysis

[0111] Graphpadprism 7.0 statistical software was used for drawing, SPSS18.0 was used for data processing, and one-way ANOVA was used for significance test for differences among multiple groups, and Student's t test was used for significance analysis for differences between two groups. Compared with the NC+LI0004 protein group, #=p<0.05 indicates significant difference, ##=p<0.01 indicates extremely significant difference; compared with the NC+LI0004 protein group and the Notch 1-siRNA group, *=p<0.05 indicates significant difference, **=p<0.01 indicates extremely significant difference.

[0112] 10. Test results

[0113] 10.1 Effect of LI0004 protein on IPEC-J2 cell activity

[0114] After the cells were treated with LI0004 protein for 24 h, LI0004 protein was detected by Western blot. Figure 1 As shown in Figure 1, 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, 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.

[0115] 10.2 Effect of LI0004 protein on the expression of Notch pathway-related proteins

[0116] 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, the Lawsonia live vaccine and 0.02, 0.04 and 0.08 mg / mL LI0004 protein treatment of IPEC-J2 cells 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.

[0117] 10.3 Effect of LI0004 protein on cell cycle

[0118] 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 expressions of cell cycle-related proteins Cyclin D1, CDK4, CDK6, and c-myc were significantly upregulated (p < 0.01).

[0119] IPEC-J2 cells were inoculated into culture dishes and treated with LI0004 protein for 24 hours before being collected for flow cytometry. 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.

[0120] 10.4 Analysis of colocalization of LI0004 protein and Notch 1 by indirect immunofluorescence double labeling assay

[0121] LI0004 protein was applied to IPEC-J2 cells, and the indirect immunofluorescence double labeling assay was used to detect the distribution of LI0004 protein in IPEC-J2 cells, the expression of Notch 1, and the co-localization analysis of the two. Figure 6As shown in the figure, the green fluorescence emitted by LI0004 protein and the red fluorescence marked Notch 1 have a highly overlapping co-localization phenomenon, and LI0004 protein is mainly distributed in the nucleus and cytoplasm. After the cells were treated with LI0004 protein, the expression level of Notch 1 also increased significantly with the increase of treatment concentration, and gradually moved to the nucleus, indicating that after LI0004 protein infected IPEC-J2 cells, it directly bound to Notch 1, increased the protein expression of Notch 1 and promoted the transfer of Notch 1 protein to the nucleus.

[0122] 10.5 Notch 1 Interference Test

[0123] Four siRNAs (Notch 1-siRNA 2513, 1928, 2291, 3591) were respectively applied to IPEC-J2 cells, and cell proteins were collected. 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.

[0124] The Notch 1 interference test was used to study the mechanism of action of LI0004 protein in inducing IPEC-J2 cell proliferation. After Notch1-siRNA transfection, Western blot was used to detect the effect of LI0004 protein on Notch pathway and cell cycle-related proteins. The results are shown in Figure 2. Figure 8 As shown in the results, interference with Notch 1 expression can significantly reduce the expression of Notch 1, Hes-1, Cyclin D3 and c-myc (p < 0.01), and LI0004 protein significantly upregulated the expression of Notch 1 and cell cycle-related proteins (p < 0.01). The results showed that Notch 1 knockdown inhibited the effect of LI0004 protein on cell proliferation.

[0125] 10.6 Co-IP analysis of the interaction between LI0004 protein and Notch 1 molecule

[0126] In order to further study the interaction between LI0004 protein and Notch 1, the present invention treated IPEC-J2 cells with purified LI0004 protein with a His tag. 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 protein was obtained by magnetic bead adsorption, and Western blot was used to detect whether Notch 1 was contained in it. The results are as follows: Fig. 9 As shown, Notch 1 antibody detected Notch 1 bands in the precipitated complex, while only weak, non-specific bands were found in the IgG control precipitated complex. The above research results indicate that LI0004 protein directly binds to Notch 1 protein, activates Notch 1 signaling pathway, and then promotes IPEC-J2 cell proliferation.

[0127] 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 infect IPEC-J2 cells as a key virulence factor of Lawsoniae.

[0128] Example 2 Effect of LI0004 protein on host cell proliferation in vivo

[0129] 1. Experimental Animals

[0130] BALB / c female mice, 4 weeks old, SPF grade, were purchased from Hubei Experimental Animal Research Center.

[0131] 2. Test methods

[0132] 2.1 Trial Grouping

[0133] 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 TCID50 / mL live vaccine of Lawsonia suis was used. The protein dosage of each mouse in the LI0004 group was 250 μg. The valnemulin was administered orally at 3 mg / kg bw per mouse. The experimental period was 14 days. After 14 days, the mice were killed and the ileum and cecum tissues of the mice were collected. The intestinal tissue samples of each segment were divided into 3 parts. One part was fixed in 10% neutral formalin solution to make histopathological sections, and the other two parts were stored in a -80℃ refrigerator for PCR detection.

[0134] 2.2 Pathological tissue testing

[0135] (1) Place mouse ileum tissue in paraformaldehyde fixative and fix for 24 hours.

[0136] (2) PBS was used to remove paraformaldehyde, and the ileum tissue was dehydrated using a dehydrator.

[0137] (3) After soaking in xylene for 40 min, soak the tissue in liquid paraffin for a long time. Use an embedding machine to embed the mouse intestinal tissue and slice it.

[0138] (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.

[0139] (5) Dewax the sections in xylene solution, 100%, 95%, 85%, and 70% ethanol.

[0140] (6) Soak the intestinal tissue slides in hematoxylin and eosin staining solutions in turn. After confirming that the staining is successful, rinse with distilled water for 1 min.

[0141] (7) Remove excess water from the intestinal tissue sections, dehydrate them in a dehydrator, and then seal the sections.

[0142] 2.3 Immunohistochemistry

[0143] (1) Dewaxing 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.

[0144] (2) Prepare a 3% hydrogen peroxide solution and place the slices in the solution at room temperature in the dark for 10 min.

[0145] (3) Use a microwave to boil the citric acid buffer, add the slices and continue heating for 2 minutes. Repeat twice (with an interval of 8 minutes between each time).

[0146] (4) Prepare 3% BSA solution and incubate the slices in the solution at room temperature for 30 min.

[0147] (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).

[0148] (6) Wash the slices three times with PBS, add rabbit IgG drops on the slices, and incubate in a 37°C incubator for 30 min (place the slices in a wet box).

[0149] (7) The treated sections were incubated in the color developing solution at room temperature and the incubation time was controlled under a microscope. The sections were washed with distilled water three times, each time for 1 min.

[0150] (8) Lightly counterstain with thiabendazole for 10 seconds and rinse with distilled water for 10 minutes for blueing.

[0151] (9) Dehydration, transparency, and sealing.

[0152] 3. Experimental results and analysis

[0153] 3.1 Mouse intestinal pathological tissue sections

[0154] After the mice were killed, the ileum tissue was collected and fixed to prepare intestinal pathological tissue sections. Fig.10 As shown in the figure, compared with the blank group, the Lawsonia live vaccine (LI) infection group showed no obvious crypt cell proliferation, while LI0004 protein infection in mice caused the intestinal villi to shorten and the intestinal wall to thicken significantly. After treatment with valnemulin, the proliferation of crypt cells and the damage to intestinal villi were alleviated, indicating that LI0004 protein can promote intestinal cell proliferation in vivo.

[0155] 3.2 Effects of Lawsonia and LI0004 proteins on protein expression in mouse intestinal tissues

[0156] 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 Lawsonia and LI0004 protein on the expression of related proteins in mouse intestinal tissues, immunohistochemical staining was used to determine the levels of Notch 1, ADAM10, c-myc and Cyclin D1 in mouse intestinal tissues. Fig.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.

[0157] From the above experimental results, it can be seen that 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 hyperplasia caused by it.

[0158] In conclusion, Notch 1 may be the key target of LI0004 protein in intestinal epithelial cell proliferation. The present invention clarifies the relationship between LI0004 protein and Notch 1 in regulating the activation of Notch signaling pathway and regulating host cell cycle at the molecular level, and preliminarily studies the mechanism of action of LI0004 protein in host cell proliferation and cell cycle toxicity caused by Lawsonia, laying a foundation for the development of anti-Lawsonia drugs targeting LI0004.

[0159] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined 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 cell proliferation; (2) Use in the preparation of medicines for treating animal diseases caused by Lawsoniae infection.

2. The use according to claim 1, characterized in that The inhibitor of the LI0004 protein includes products that knock down or inhibit the expression of Notch1 to reduce the binding of the Notch1 protein to the LI0004 protein.

3. The use according to claim 2, characterized in that The product for knocking down or inhibiting Notch1 expression includes 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.

4. The use according to claim 3, characterized in that The sense strand and antisense strand of the siRNA are shown in SEQ ID NO: 5-6.

5. The use according to claim 1, characterized in that The cells include IPEC-J2 cells.

6. Application of LI0004 protein target in the preparation of drugs for inhibiting the proliferation of Lawsonia infected cells.

7. Application of LI0004 protein in preparing cell models or animal models of cell proliferation.

8. The use according to claim 7, characterized in that The cell proliferation cell model or animal model is constructed by infecting IPEC-J2 cells or animals with LI0004 protein.

9. The use according to claim 8, characterized in that The infection concentration of the LI0004 protein is 0.02-0.08 mg / mL.

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