Porcine epidemic diarrhea resistance-related gene CLDN4 and application of encoding protein thereof

Abstract

The invention discloses a porcine epidemic diarrhea resistance-related gene CLDN4 and application of a coding protein thereof, and belongs to the technical field of gene engineering. Porcine epidemic diarrhea disease resistance related gene CLDN4 in a Vero-E6 cell is respectively knocked down and overexpressed, then the Vero-E6 is infected with the porcine epidemic diarrhea virus at different infection times and infection complex numbers, and detection shows that when the CLDN4 in the Vero-E6 is knocked down, when the Vero-E6 is infected with the porcine epidemic diarrhea virus at different time points and after the Vero-E6 is infected with the different infection complex numbers, the CLDN4 in the Vero-E6 is knocked down, the CLDN4 in the Vero-E6 is knocked down. The expression level of mRNA and N protein of the M gene of the porcine epidemic diarrhea virus is obviously improved; on the contrary, when the expression quantity of the CLDN4 in the Vero-E6 is increased, the expression levels of mRNA and N protein of the M gene of the porcine epidemic diarrhea virus are obviously reduced. The invention provides a new direction and method for preventing and controlling porcine epidemic diarrhea and assisting molecular breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly to the application of the porcine epidemic diarrhea resistance-related gene CLDN4 and its encoded protein. Background Art

[0002] Porcine epidemic diarrhea virus (PEDV) belongs to the genus Alphacoronavirus of the Coronaviridae family. This virus can cause porcine epidemic diarrhea (PED), which is an acute and highly contagious intestinal disease with a lethality rate of up to 80 - 100% in neonatal piglets. Since PEDV was discovered in the 1970s, it has caused multiple large-scale epidemics globally. Due to the continuous emergence of recombinant or newly isolated PEDV strains in recent years, PEDV shows a highly complex variability, greatly reducing the effectiveness of vaccine immunity and drug treatment against PEDV. Therefore, improving the resistance of pigs to epidemic diarrhea disease at the genetic level has become another important breakthrough. Identifying and analyzing the functions and mechanisms of PEDV resistance genes, assisting in molecular disease-resistant breeding, and fundamentally preventing and controlling the occurrence of PEDV have important theoretical significance and practical value for the healthy and sustainable development of the pig-raising industry in China. Summary of the Invention

[0003] The purpose of the present invention is to provide the application of the porcine epidemic diarrhea resistance-related gene CLDN4 and its encoded protein to solve the problems existing in the above-mentioned prior art. By overexpressing claudin-4 in Vero-E6, the mRNA expression level of the M gene and the N protein expression level of porcine epidemic diarrhea virus are significantly reduced, and the resistance to porcine epidemic diarrhea disease can be significantly improved.

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

[0005] The present invention provides the application of the porcine epidemic diarrhea resistance-related gene CLDN4 in any one of the following:

[0006] (1) Application in pig breeding against porcine epidemic diarrhea virus;

[0007] (2) Application in the preparation of drugs for improving the resistance to porcine epidemic diarrhea disease;

[0008] (3) Application in the preparation of drugs for preventing and controlling porcine epidemic diarrhea virus infection.

[0009] The present invention also provides the application of the protein encoded by the porcine epidemic diarrhea resistance-related gene CLDN4 in any one of the following:

[0010] (1) Application in pig breeding against porcine epidemic diarrhea virus;

[0011] (2) Use in the preparation of a drug for enhancing resistance to porcine epidemic diarrhea;

[0012] (3) Use in the preparation of a drug for preventing and controlling porcine epidemic diarrhea virus infection.

[0013] The present invention also provides the use of a recombinant vector containing the porcine epidemic diarrhea disease-resistant related gene CLDN4 in any of the following:

[0014] (1) Use in the breeding of pigs resistant to porcine epidemic diarrhea virus;

[0015] (2) Use in the preparation of a drug for enhancing resistance to porcine epidemic diarrhea;

[0016] (3) Use in the preparation of a drug for preventing and controlling porcine epidemic diarrhea virus infection;

[0017] Wherein, the recombinant vector is obtained by ligating the porcine epidemic diarrhea disease-resistant related gene CLDN4 with an expression vector.

[0018] The present invention also provides the use of a host bacterium containing the recombinant vector in any of the following:

[0019] (1) Use in the breeding of pigs resistant to porcine epidemic diarrhea virus;

[0020] (2) Use in the preparation of a drug for enhancing resistance to porcine epidemic diarrhea;

[0021] (3) Use in the preparation of a drug for preventing and controlling porcine epidemic diarrhea virus infection;

[0022] The recombinant vector is obtained by ligating the porcine epidemic diarrhea disease-resistant related gene CLDN4 with an expression vector, and the porcine epidemic diarrhea disease-resistant related gene CLDN4 is integrated into the genome of the host bacterium through the recombinant vector.

[0023] Optionally, a pig breed with enhanced resistance to porcine epidemic diarrhea virus is obtained by increasing the expression level of CLDN4.

[0024] The present invention also provides the use of a reagent for detecting the expression level of the porcine epidemic diarrhea disease-resistant related gene CLDN4 in the preparation of a detection kit for preventing and controlling porcine epidemic diarrhea virus infection.

[0025] The present invention also provides the use of the porcine epidemic diarrhea disease-resistant related gene CLDN4 in the preparation of a cell model with enhanced resistance to porcine epidemic diarrhea virus infection. By overexpressing the CLDN4 in cells, the resistance of pigs to porcine epidemic diarrhea virus infection is enhanced. The gene accession number of CLDN4 in the NCBI database is XM_008018331.2.

[0026] The present invention also provides a method for preventing and controlling porcine epidemic diarrhea disease for non-therapeutic purposes in vitro, which includes the step of overexpressing the porcine epidemic diarrhea disease-resistant related gene CLDN4 in porcine recipient cells.

[0027] The present invention also provides a method for breeding porcine breeds resistant to porcine epidemic diarrhea disease, which includes the steps of overexpressing the porcine epidemic diarrhea disease-resistant related gene CLDN4 in porcine recipient cells and retaining porcine individuals with significantly reduced expression levels of the M gene and N protein of porcine epidemic diarrhea virus.

[0028] The present invention discloses the following technical effects:

[0029] The present invention constructs siRNA and overexpression vectors for CLDN4, and respectively knocks down and overexpresses the expression level of CLDN4 in Vero-E6 cells through transfection technology; then PEDV infects Vero-E6 at different infection times (hpi) and multiplicities of infection (MOI), and detects the influence of changes in the CLDN4 expression level on PEDV infection. The results show that when CLDN4 in Vero-E6 is knocked down, after PEDV infects Vero-E6 at different time points and with different MOI, the mRNA expression level of the PEDV M gene and the expression level of the N protein are significantly increased; conversely, when the expression level of CLDN4 in Vero-E6 is increased, the mRNA expression level of the PEDV M gene and the expression level of the N protein are significantly decreased. This shows that knocking down CLDN4 in Vero-E6 can improve the ability to infect PEDV, and conversely, overexpressing CLDN4 in Vero-E6 reduces the ability to infect PEDV. The present invention provides a new direction and method for preventing and controlling PEDV and assisting in molecular disease-resistant breeding work. Description of the Drawings

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the following described drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0031] Figure 1 Shows the influence of different time points (A) after PEDV infection and different MOI infection conditions (B) on the mRNA expression level of the PEDV M gene in Vero-E6 after knocking down CLDN4 under different treatment conditions in the present invention;

[0032] Figure 2The effects of the knockdown of CLDN4 in Vero-E6 under different treatment conditions on the expression level of PEDV N protein at different time points (A) and under different MOI infection conditions (B) after PEDV infection in the present invention;

[0033] Figure 3 The effects of the overexpression of CLDN4 in Vero-E6 under different treatment conditions on the expression level of PEDV M mRNA at different time points (A) and under different MOI infection conditions (B) after PEDV infection in the present invention;

[0034] Figure 4 The effects of the overexpression of CLDN4 in Vero-E6 on the expression level of PEDV N protein at different time points after PEDV infection in the present invention;

[0035] Figure 5 The effects of the overexpression of CLDN4 in Vero-E6 on the expression level of PEDV N protein under different MOI PEDV infections in the present invention. Detailed implementation manners

[0036] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0037] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0038] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can 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 related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0039] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the specification of the present invention, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0040] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0041] Example 1

[0042] 1. Test materials

[0043] 1.1 Cells, viruses, vectors

[0044] Vero-E6 was purchased from the Cell Bank of the Chinese Academy of Sciences Committee for Type Culture Collection, catalog number: GNO17; the classical strain CV777 of PEDV was preserved in this laboratory; pcDNA3.1(+) was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0045] 1.2 Main reagents

[0046] (1) First-Strand cDNA Synthesis Super Mix (catalog number: AE301-02; TransGen Biotech);

[0047] (2) High Fidelity (HiFi) PCR SuperMix II (-dye) (catalog number: AS131-21; TransGen Biotech);

[0048] (3) Lipofectamine RNAiMAX transfection reagent (catalog number: 13778150; invitrogen);

[0049] (4) Lipofectamine TM 3000 reagent (catalog number: L3000015; invitrogen)

[0050] (5) PEDV N antibody (catalog number: JN1401; MEDIAN);

[0051] (6) Alexa Fluor 488-labeled goat anti-mouse IgG (H+L) (catalog number: A0428; Beyotime).

[0052] 2. Test methods

[0053] 2.1 Cell culture

[0054] Take Vero-E6 cells cryopreserved in liquid nitrogen and quickly thaw them in a 37 °C water bath. Transfer the cell suspension to a 15 mL centrifuge tube, add an appropriate amount of complete medium (90% DMEM medium + 10% fetal bovine serum), and gently pipette to mix well. After mixing, centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the cells, and then plate the cells. The cells are placed in an incubator at a temperature of 37 °C, humidity of 95%, and 5% CO 2 and cultured. When the cell density reaches over 95%, passage the cells. Discard the medium, wash twice with PBS, add 1 mL of 0.25% trypsin and digest for 2 - 3 min, then add 3 mL of complete medium to terminate the digestion. Collect the digested cells in a centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend with complete medium, and then perform passage culture.

[0055] 2.2 Establishment of PEDV-infected Vero-E6 cell model

[0056] Take Vero-E6 cells that have been passaged twice after resuscitation and evenly plate them in a 6-well cell culture plate. When they grow to about 95%, infect Vero-E6 with PEDV at the corresponding multiplicity of infection. The method is as follows: Discard the culture medium, wash three times with PBS, add the corresponding amount of PEDV virus solution, incubate in a 37 °C incubator for 1.5 h, and shake the cell culture plate every 30 min. After incubation, discard the virus solution, wash three times with PBS, add 2 mL of DMEM medium, and continue to culture. Collect the cells at the corresponding infection time for the next detection.

[0057] 2.3 siRNA design and transfection

[0058] The siRNA of CLDN4 (accession number: XM_008018331.2) used in this invention was designed by the online design tool of Thermo Fisher Scientific (https: / / rnaidesigner.thermofisher.com / rnaiexpress / ) and synthesized by Suzhou GeneCreate Biotech Co., Ltd. The specific siRNA sequences are shown in the table:

[0059] Table 1 siRNA sequences

[0060]

[0061] For the cell siRNA transfection experiment, Lipofectamine RNAiMAX transfection reagent was used. The specific transfection steps are as follows:

[0062] (1) Inoculate cells with good growth status into a 6-well plate and perform transfection when the cells reach about 60 - 80%.

[0063] (2) Prepare 2 sterile EP tubes, labeled as A and B, add opti-MEM at a volume of 125 μL per well. Add siRNA (25 pmol per well) to tube A, and add RNAiMAX transfection reagent at a volume of 7.5 μL per well to tube B. Gently pipette to mix the solutions in tubes A and B respectively, then mix the solutions in tubes A and B together and gently mix well. Incubate at room temperature for 5 min.

[0064] (3) After incubation, add the siRNA / RNAiMAX complex at 250 μL per well.

[0065] (4) Conduct the PEDV-infected cell experiment 24 h after transfection.

[0066] 2.4 Construction of gene overexpression vector

[0067] Extract the RNA from the Vero-E6 cell line and use the TransGen Biotech PCR reaction kit First-Strand cDNA Synthesis Super Mix to perform reverse transcription to obtain cDNA.

[0068] Table 2 First-strand cDNA synthesis

[0069]

[0070] Reaction system: 42 °C for 30 min, 85 °C for 5 s.

[0071] Use TransGen Biotech High Fidelity (HiFi) PCR SuperMix II (-dye) to amplify the CDS region of the target gene. The specific PCR reaction system is as follows:

[0072] Table 3 PCR reaction system

[0073]

[0074]

[0075] The reaction program is set according to the following conditions: 94 °C for 3 min; 94 °C for 30 s, 58 °C for 30 s, 72 °C for 1 min, 35 cycles; 72 °C for 10 min.

[0076] Forward Primer (SEQ ID NO.5): 5’-CAAGCTTGCCACCATGGCCTCCATGGGGCTACA-3’;

[0077] R Primer (SEQ ID NO.6): 5’-CCGGAATTCTTACACGTAGTTGCTGGCAGCAGC-3’.

[0078] The pcDNA3.1(+) plasmid was digested with HindIII / EcoRI. The digestion system used was as follows:

[0079] Table 4 Digestion reaction system

[0080]

[0081] Reaction conditions: 37°C, 1 h.

[0082] After linearizing the pcDNA3.1(+) vector, the CLDN4 amplification product and the linearized pcDNA3.1(+) were purified using a gel extraction kit. Then, In-Fusion Snap Assembly Master Mix was used for ligation. The ligation reaction system is shown in Table 5 below. Then, it was transferred to DH5α competent cells. Single colonies after shaking the bacteria were selected to determine their positive rate. After sequencing and plasmid extraction, the positive plasmid was finally obtained.

[0083] Table 5 Ligation reaction

[0084]

[0085] 2.5 RNA extraction, reverse transcription, fluorescence quantitative PCR

[0086] Total cellular RNA was extracted using Trizol. The specific steps were as follows:

[0087] (1) Prepare a 4°C centrifuge in advance. Gently wash the cells with PBS 3 times. Add 1 mL of Trizol to each bottle, pipette up and down repeatedly, then collect the liquid into a 1.5 mL imported sterile and enzyme-free centrifuge tube. After shaking and mixing well, let it stand for 5 min;

[0088] (2) Add 200 μL of chloroform, shake and mix well, let it stand for 3 min until the liquid layers. Then, centrifuge at 4°C, 12000 g for 15 min;

[0089] (3) Pipette the upper colorless liquid phase into a new sterile and enzyme-free 1.5 mL centrifuge tube, add an equal volume of isopropanol, invert and mix well, then place it at -20°C for 30 min;

[0090] (4) Centrifuge at 12000 g for 10 min at 4°C and discard the supernatant;

[0091] (5) Add 1 mL of pre-cooled 75% ethanol in advance, pipette up and down until the precipitate floats, then centrifuge at 7500 g for 5 min at 4°C;

[0092] (6) Carefully remove the supernatant, and leave it at room temperature until the precipitate dries and becomes transparent;

[0093] (7) Add 30 μL of nuclease-free water and incubate at room temperature for 10 min.

[0094] (8) Use the Agilent Bioanalyzer 2100 to detect the integrity (RIN value) of the sample; and further examine the band distribution by 1% agarose gel electrophoresis, and use NanoDrop to detect the quality and concentration of RNA.

[0095] The RNA samples with qualified quality detection are used for reverse transcription reaction, and the following operations are carried out according to the operation instructions of the reverse transcription kit PrimeScript TM RT reagent Kit with gDNA Eraser:

[0096] (1) Remove the potential genomic DNA contamination in the sample. The system is shown in Table 6, and the reaction condition is 8 min at room temperature. After completion, proceed to the next step.

[0097] Table 6 Reaction system for removing genomic contamination

[0098]

[0099] (2) The reverse transcription reaction system is shown in Table 7, and the reaction conditions are 15 min at 37 °C and 5 s at 85 °C.

[0100] Table 7 Reverse transcription reaction system

[0101]

[0102]

[0103] After reverse transcription into cDNA, real-time fluorescence quantitative PCR is carried out. The PCR reaction system is 10 μL: 5 μL of SYBP Green Real-time PCR MasterMix, 0.5 μL each of the upstream and downstream primers (10 μmol / L), 1 μL of cDNA template, and 3 μL of ddH 2 O. The primers are shown in Table 8 below. Each sample is repeated 3 times, with the GAPDH gene as the internal reference, and the relative expression level of PEDV M gene mRNA is analyzed using 2 -△△Ct to analyze the relative expression level of PEDV M gene mRNA.

[0104] Table 8 Primer information

[0105]

[0106] 2.6 Western blot

[0107] (1) Preparation of total cellular proteins: Prepare a protein extraction working solution by mixing RIPA (1 mL) and PMSF (10 μL) in a ratio of 1 mL:10 μL. Add an appropriate amount of the working solution to the cells, place on ice for 10 min for lysis, collect the lysed cells in a sterile EP tube, centrifuge at 12,000 g / min at 4 °C for 15 min, and take the supernatant for subsequent experiments.

[0108] (2) Protein concentration determination: Determine the protein concentration according to the instructions of the BCA protein quantification kit.

[0109] (3) Denaturation of protein samples: Mix the protein supernatant and 5× protein loading buffer in a ratio of 4:1. Denature the mixed protein solution at 95 °C for 5 min, quickly place on ice for 1 min, and store at -20 °C for later use.

[0110] (4) Sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis: Take the denatured protein samples above, add 30 μg of the sample to be tested to each well. Apply a constant voltage of 80 V for the stacking gel. After the marker is separated, change the voltage to 120 V for the separating gel. Run the electrophoresis for about 1 h until the bromophenol blue dye reaches the bottom of the separating gel to end the electrophoresis.

[0111] (5) Transfer: Take out the PAGE gel, and prepare a black filter, filter paper, PAGE gel, PVDF membrane, filter paper, and black filter electrotransfer "sandwich" in an ice box for membrane transfer. The membrane transfer conditions are a constant voltage of 110 V for 30 min.

[0112] (6) Blocking: After membrane transfer, place the PVDF membrane in the blocking solution and block it on a shaker at room temperature for 2 h.

[0113] (7) Primary antibody incubation: Dilute the primary antibody according to the appropriate ratio. Take out the PVDF membrane and rinse it 3 times in 1×TBST for 10 min each time, then transfer it to the primary antibody dilution solution and incubate overnight at 4 °C.

[0114] (8) Secondary antibody incubation: Transfer the PVDF membrane to a decolorizing box, add an appropriate amount of 1×TBST, and rinse it on a shaker at room temperature 3 times for 10 min each time. Select the corresponding species secondary antibody according to the source of the primary antibody and dilute it according to the instructions. Transfer the PVDF membrane obtained from the previous step to the secondary antibody incubation solution and incubate at room temperature for about 1 h.

[0115] (9) Development and exposure: After secondary antibody incubation, rinse the PVDF membrane, prepare the developing solution, and use Image Lab for development and exposure to observe the protein bands.

[0116] 2.7 Indirect immunofluorescence

[0117] (1) Collect the relevant processed cell samples, wash them 3 times with PBS, and fix them with a fixing solution containing 4% paraformaldehyde at room temperature for 20 min.

[0118] (2) Discard the fixing solution, wash 3 times with PBS, 5 min each time; permeabilize with 0.2% Triton at room temperature for 10 min.

[0119] (3) Discard the permeabilizing solution, wash 3 times with PBS, 5 min each time; block with 5% BSA at room temperature for 30 min.

[0120] (4) Discard the blocking solution, incubate with PEDV N protein antibody (1:300) dilution, overnight at 4 °C.

[0121] (5) After the primary antibody blocking is completed, wash 3 times with PBS, add the secondary antibody with fluorescent label (1:300 dilution), and incubate in the dark at room temperature for 1 h.

[0122] (6) After the secondary antibody incubation is completed, wash 3 times with PBS, add DAPI to stain the cell nuclei, and incubate in the dark at room temperature for 5 min.

[0123] (7) After the nuclear staining is completed, wash 3 times with PBS, mount with an anti-fluorescence quencher, and observe and photograph with a confocal microscope.

[0124] 3. Results

[0125] After infecting cells with different infection times and different MOIs, collect cell RNA samples and detect the mRNA expression level of the viral M gene by q-PCR. As Figure 1 shown, the results show that after CLDN4 knockdown, the expression level of the viral M gene is significantly increased under different infection times (12 h, 24 h, and 36 h) and different MOIs (0.01, 0.05, and 0.1 MOI) infection conditions, that is, the decrease in CLDN4 expression level can promote PEDV infection.

[0126] After infecting cells with different infection times and different MOIs, collect cell protein samples and detect the expression level of the viral PEDV N protein by Western blot technology. As Figure 2 shown, the results show that after CLDN4 knockdown, the expression level of PEDV N protein is significantly increased under different infection times (12 h, 24 h, and 36 h) and different MOIs (0.01, 0.05, and 0.1 MOI) infection conditions, that is, the decrease in CLDN4 expression level can promote PEDV infection.

[0127] At different time points after PEDV infection and with different MOIs infecting cells, collect cell RNA samples and detect the mRNA expression level of the viral M gene by q-PCR. As Figure 3As shown, the results showed that after CLDN4 overexpression, the expression levels of the viral M gene were significantly decreased at various time points of infection (12 h, 24 h, and 36 h) and under different MOIs (0.01, 0.05, and 0.1 MOI), that is, an increase in the CLDN4 expression level could inhibit PEDV infection.

[0128] Cells were infected with PEDV at different time points and with different MOIs, and cell protein samples and cell smear samples were collected. The expression levels of the viral PEDV N protein were detected by Western blot and indirect immunofluorescence techniques, respectively. As Figure 4 and Figure 5 shown, the results showed that after CLDN4 overexpression, the expression levels of the PEDV N protein were significantly decreased at different infection times (12 h, 24 h, and 36 h) and under different MOIs (0.01, 0.05, and 0.1 MOI), that is, an increase in the CLDN4 expression level could inhibit PEDV infection.

[0129] The above-described embodiments are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of porcine epidemic diarrhea resistance-related gene CLDN4 in any of the following: (1) Application in pig breeding for resistance to porcine epidemic diarrhea virus; (2) Application in the preparation of drugs for improving resistance to porcine epidemic diarrhea; (3) Application in the preparation of drugs for preventing and controlling porcine epidemic diarrhea virus infection.

2. Application of the protein encoded by the porcine epidemic diarrhea resistance-related gene CLDN4 in any of the following: (1) Application in pig breeding for resistance to porcine epidemic diarrhea virus; (2) Application in the preparation of drugs for improving resistance to porcine epidemic diarrhea; (3) Application in the preparation of drugs for preventing and controlling porcine epidemic diarrhea virus infection.

3. Use of a recombinant vector comprising porcine epidemic diarrhea resistance-related gene CLDN4 in any of the following: (1) Application in pig breeding for resistance to porcine epidemic diarrhea virus; (2) Application in the preparation of drugs for improving resistance to porcine epidemic diarrhea; (3) Application in the preparation of drugs for preventing and controlling porcine epidemic diarrhea virus infection; in, The recombinant vector is obtained by connecting the porcine epidemic diarrhea resistance-related gene CLDN4 with an expression vector.

4. Use of a host bacterium containing a recombinant vector in any of the following: (1) Application in pig breeding for resistance to porcine epidemic diarrhea virus; (2) Application in the preparation of drugs for improving resistance to porcine epidemic diarrhea; (3) Application in the preparation of drugs for preventing and controlling porcine epidemic diarrhea virus infection; The recombinant vector is obtained by connecting the porcine epidemic diarrhea resistance-related gene CLDN4 with an expression vector, and the porcine epidemic diarrhea resistance-related gene CLDN4 is integrated into the genome of the host bacteria through the recombinant vector.

5. The use according to any one of claims 1 to 4, characterized in that: A pig breed with improved resistance to porcine epidemic diarrhea virus is obtained by increasing the expression level of the CLDN4.

6. Use of a reagent for detecting the expression level of porcine epidemic diarrhea resistance-related gene CLDN4 in the preparation of a detection kit for resistance to porcine epidemic diarrhea virus infection.

7. Use of porcine epidemic diarrhea resistance-related gene CLDN4 in preparing a cell model for improving resistance to porcine epidemic diarrhea virus infection, characterized in that: By overexpressing the CLDN4 in cells, the resistance of pigs to epidemic diarrhea virus infection is improved. The gene accession number of the CLDN4 in the NCBI database is XM_008018331.

2.

8. An in vitro non-therapeutic method for preventing and controlling porcine epidemic diarrhea, characterized in that: The method comprises the steps of overexpressing porcine epidemic diarrhea resistance-related gene CLDN4 in pig recipient cells.

9. A method for breeding pigs resistant to porcine epidemic diarrhea, characterized in that: The method comprises the steps of overexpressing the porcine epidemic diarrhea resistance-related gene CLDN4 in porcine recipient cells and retaining pig individuals with significantly reduced expression levels of the porcine epidemic diarrhea virus M gene and N protein.

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