Application of porcine epidemic diarrhea resistance-related gene CLDN4 and its encoded protein

Abstract

The present invention discloses the application of porcine epidemic diarrhea resistance-related gene CLDN4 and its encoded protein, which belongs to the field of genetic engineering technology. The present invention knocks down and overexpresses porcine epidemic diarrhea resistance-related gene CLDN4 in Vero-E6 cells, and then uses porcine epidemic diarrhea virus to infect Vero-E6 at different infection times and multiplicity of infection. It is found by detection that: when CLDN4 in Vero-E6 is knocked down, at different time points of porcine epidemic diarrhea virus infection and after infecting Vero-E6 at different multiplicity of infection, the expression levels of mRNA and N protein of porcine epidemic diarrhea virus M gene are significantly increased; conversely, when CLDN4 expression in Vero-E6 increases, the expression levels of mRNA and N protein of porcine epidemic diarrhea virus M gene are significantly reduced. The present invention provides new directions and methods for preventing and controlling porcine epidemic diarrhea and assisting molecular breeding.

Description

Technical Field

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

[0002] Porcine epidemic diarrhea virus (PEDV) belongs to the genus Alphacoronavirus in the family Coronaviridae. This virus causes porcine epidemic diarrhea (PED), an acute, highly contagious intestinal disease with a mortality rate of 80-100% in newborn piglets. Since its discovery in the 1970s, PEDV has caused numerous large-scale epidemics worldwide. Due to the continuous emergence of recombinant or newly isolated PEDV strains in recent years, PEDV exhibits highly complex variability, significantly reducing the effectiveness of vaccines and drug treatments against PEDV. Therefore, improving pigs' genetic resistance to PEDV has become another important breakthrough. Identifying and analyzing the functions and mechanisms of PEDV-resistant genes, assisting molecular disease-resistant breeding, and fundamentally preventing and controlling the occurrence of PEDV have important theoretical and practical significance for the healthy and sustainable development of my country's pig industry. 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 tight junction protein 4 in Vero-E6, the expression levels of the mRNA and N protein of the porcine epidemic diarrhea virus M gene are significantly reduced, which can significantly improve the resistance to porcine epidemic diarrhea.

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

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

[0006] (1) Application in pig breeding for resistance to porcine epidemic diarrhea virus;

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

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

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

[0010] (1) Application in pig breeding for resistance to porcine epidemic diarrhea virus;

[0011] (2) Application in the preparation of drugs for improving resistance to porcine epidemic diarrhea;

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

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

[0014] (1) Application in pig breeding for resistance to porcine epidemic diarrhea virus;

[0015] (2) Application in the preparation of drugs for improving resistance to porcine epidemic diarrhea;

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

[0017] The recombinant vector is obtained by connecting the porcine epidemic diarrhea resistance-related gene CLDN4 with an expression vector.

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

[0019] (1) Application in pig breeding for resistance to porcine epidemic diarrhea virus;

[0020] (2) Application in the preparation of drugs for improving resistance to porcine epidemic diarrhea;

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

[0022] 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.

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

[0024] The present invention also provides use of a reagent for detecting the expression level of porcine epidemic diarrhea resistance-related gene CLDN4 in preparing a detection kit for resisting porcine epidemic diarrhea virus infection.

[0025] The present invention also provides the use of the porcine epidemic diarrhea resistance-related gene CLDN4 in preparing a cell model for improving resistance to porcine epidemic diarrhea virus infection. By overexpressing the CLDN4 in the cells, the pig's resistance to epidemic diarrhea virus infection is improved. The gene accession number of the CLDN4 in the NCBI database is XM_008018331.2.

[0026] The present invention also provides an in vitro non-therapeutic method for preventing and controlling porcine epidemic diarrhea, comprising the step of overexpressing the porcine epidemic diarrhea resistance-related gene CLDN4 in porcine recipient cells.

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

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

[0029] The present invention constructs siRNA and overexpression vectors for CLDN4, and knocks down and overexpresses the expression level of CLDN4 in Vero-E6 cells by transfection technology; then PEDV infects Vero-E6 at different infection times (hpi) and multiplicity of infection (MOI), and detects the effect of changes in CLDN4 expression levels on PEDV infection. The results show that when CLDN4 is knocked down in Vero-E6, the expression levels of PEDV M gene mRNA and N protein are significantly increased at different time points of PEDV infection and after Vero-E6 is infected with different MOIs; conversely, when the expression level of CLDN4 in Vero-E6 is increased, the expression levels of PEDV M gene mRNA and 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 new directions and methods for preventing and controlling PEDV and assisting molecular disease resistance breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] 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.

[0031] Figure 1 The effects of knockdown of CLDN4 in Vero-E6 under different treatment conditions in the present invention on the expression level of PEDV M gene mRNA at different time points (A) and at different MOI infection conditions (B);

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

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

[0034] Figure 4 The effect of 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 This is the effect of different MOIPEDV infections on the expression level of PEDV N protein after CLDN4 is overexpressed in Vero-E6 in the present invention. DETAILED DESCRIPTION

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0041] Example 1

[0042] 1. Test materials

[0043] 1.1 Cells, viruses, and vectors

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

[0045] 1.2 Main Reagents

[0046] (1) First-Strand cDNA Synthesis Super Mix (Cat. No. AE301-02; Full Formula Gold);

[0047] (2) High Fidelity (HiFi) PCR SuperMix II (-dye) (Cat. No.: AS131-21; all gold);

[0048] (3) Lipofectamine RNAiMAX transfection reagent (Cat. No. 13778150; Invitrogen);

[0049] (4)Lipofectamine TM 3000 reagent (Cat. No. L3000015; Invitrogen)

[0050] (5) PEDV N antibody (Cat. No.: JN1401; MEDIAN);

[0051] (6) Alexa Fluor 488-labeled goat anti-mouse IgG (H+L) (Cat. No. A0428; Beyotime).

[0052] 2. Test methods

[0053] 2.1 Cell culture

[0054] Take the Vero-E6 frozen in liquid nitrogen and thaw it quickly in a 37°C water bath. Transfer the cell suspension to a 15mL centrifuge tube, add an appropriate amount of complete culture medium (90% DMEM culture medium + 10% fetal bovine serum) and gently blow to mix. After mixing, centrifuge at 1000rpm for 5 minutes, discard the supernatant, resuspend the cells and plate the cells. Culture the cells in a constant temperature incubator with a temperature of 37°C, a humidity of 95%, and 5% CO2. When the cell density reaches more than 95%, subculture is carried out, the culture medium is discarded, and PBS is washed twice. 1mL of 0.25% trypsin is added for digestion for 2-3 minutes, and 3mL of complete culture medium is added to terminate the digestion. The digested cells are collected in a centrifuge tube and centrifuged at 1000rpm for 5 minutes, the supernatant is discarded, and the cells are resuspended in complete culture medium and then subcultured.

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

[0056] Vero-E6 cells, passaged twice after thawing, were evenly plated in 6-well cell culture plates. When the cells reached approximately 95% growth, they were infected with PEDV at the appropriate multiplicity of infection (MOI). The culture medium was discarded, the cells were washed three times with PBS, and the appropriate amount of PEDV virus solution was added. The cells were incubated in a 37°C incubator for 1.5 hours, with the cell culture plate shaken every 30 minutes. After incubation, the virus solution was discarded, the cells were washed three times with PBS, and 2 mL of DMEM medium was added for further culture. At the appropriate infection time, the cells were harvested for further testing.

[0057] 2.3siRNA design and transfection

[0058] The siRNA for CLDN4 (accession number: XM_008018331.2) used in the present invention was designed using the ThermoFisherscientific online design tool (https: / / rnaidesigner.thermofisher.com / rnaiexpress / ) and synthesized by Suzhou GeneWeizhi Biotechnology Co., Ltd. The specific siRNA sequences are shown in the table:

[0059] Table 1 siRNA sequences

[0060]

[0061] Lipofectamine RNAiMAX transfection reagent was used for cell siRNA transfection experiments. The specific transfection steps are as follows:

[0062] (1) Cells in good growth condition were seeded into a six-well plate and transfected when the cells reached about 60-80%.

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

[0064] (3) After incubation, add 250 μL of siRNA / RNAiMAX complex to each well.

[0065] (4) PEDV infection cell experiment was performed 24 h after transfection.

[0066] 2.4 Construction of gene overexpression vector

[0067] RNA was extracted from Vero-E6 cells and PCR reaction was performed using the full-scale gold PCR kit. cDNA was obtained by reverse transcription using First-Strand cDNA Synthesis Super Mix.

[0068] Table 2 First-strand cDNA synthesis

[0069]

[0070] Reaction system: 42℃30min, 85℃5s.

[0071] Using full gold High Fidelity (HiFi) PCR SuperMix II (-dye) was used 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 was 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] F Primer (SEQ ID NO.5): 5'-CAAGCTTGCCACCATGGCCTCCATGGGGCTACA-3';

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

[0078] The pcDNA3.1(+) plasmid was double-digested with HindIII / EcoRI using the following enzyme digestion system:

[0079] Table 4 Enzyme digestion reaction system

[0080]

[0081] Reaction conditions: 37°C, 1h.

[0082] After linearization of the pcDNA3.1(+) vector, the CLDN4 amplified product and the linearized pcDNA3.1(+) were purified using a gel extraction kit. Ligation was then performed using the In-Fusion Snap Assembly Master Mix. The ligation reaction system is shown in Table 5 below. The cells were then transformed into DH5α competent cells. Single colonies were selected after shaking and their positive rates were determined. Positive plasmids were finally obtained through sequencing and plasmid extraction.

[0083] Table 5 Ligation reaction

[0084]

[0085] 2.5 RNA extraction, reverse transcription, and fluorescent quantitative PCR

[0086] Total RNA was extracted from cells using Trizol. The specific steps are as follows:

[0087] (1) Prepare a 4°C centrifuge in advance, gently wash the cells 3 times with PBS, add 1 mL of Trizol to each bottle, pipette repeatedly, collect the liquid into a 1.5 mL imported sterile enzyme-free centrifuge tube, shake and mix, and let it stand for 5 minutes;

[0088] (2) Add 200 μL of chloroform, shake and mix, let stand for 3 min until the liquid separates, and then centrifuge at 12,000 g at 4°C for 15 min;

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

[0090] (4) Centrifuge at 12,000 g for 10 min at 4°C and discard the supernatant;

[0091] (5) Add 1 mL of pre-chilled 75% ethanol, invert until the precipitate floats, and centrifuge at 7500 g for 5 min at 4°C.

[0092] (6) Carefully remove the supernatant and place at room temperature until the precipitate becomes dry and transparent;

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

[0094] (8) The integrity of the samples (RIN value) was tested using an Agilent Bioanalyzer 2100. The band distribution was examined by 1% agarose gel electrophoresis, and the RNA quality and concentration were tested by NanoDrop.

[0095] RNA samples that passed the quality test were used for reverse transcription reaction according to the reverse transcription kit PrimeScript TM RT reagent Kit with gDNA Eraser operating instructions:

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

[0097] Table 6 Genomic contamination removal reaction system

[0098]

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

[0100] Table 7 Reverse transcription reaction system

[0101]

[0102]

[0103] After reverse transcription into cDNA, real-time fluorescence quantitative PCR was performed. The PCR reaction system was 10 μL: SYBP Green Real-time PCR Master Mix 5 μL, 0.5 μL each of upstream and downstream primers (10 μmol / L), 1 μL cDNA template, and 3 μL ddH2O. The primers are shown in Table 8 below. Each sample was repeated 3 times, with GAPDH gene as the internal reference and 2 -△△Ct The relative expression level of PEDV M gene mRNA was analyzed.

[0104] Table 8 Primer information

[0105]

[0106] 2.6 Western blot

[0107] (1) Preparation of total cell protein: Prepare protein extraction working solution according to the ratio of RIPA 1 mL: protease inhibitor (PMSF) 10 μL. Add appropriate amount of working solution to cells and place on ice for 10 min. Collect the lysed cells in a sterile EP tube and centrifuge at 12000 g / min at 4 °C for 15 min. Take the supernatant for subsequent experiments.

[0108] (2) Protein concentration determination: Protein concentration was determined 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 sample mentioned above, add 30 μg of the sample to be tested to each well, and run the concentrated gel at a constant voltage of 80 V. After the marker is separated, the voltage can be changed to 120 V and the separation gel is run. After about 1 hour of electrophoresis, the electrophoresis is terminated when the bromophenol blue dye reaches the bottom of the separation gel.

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

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

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

[0114] (8) Secondary antibody incubation: Transfer the PVDF membrane to a decolorization box, add an appropriate amount of 1×TBST, and rinse on a shaker at room temperature three times, 10 minutes each time. Select the secondary antibody corresponding to the species of the primary antibody and dilute it according to the ratio in 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 hour.

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

[0116] 2.7 Indirect immunofluorescence

[0117] (1) Collect the cell samples after relevant treatment, wash them three times with PBS, and fix them with a fixative containing 4% paraformaldehyde at room temperature for 20 minutes.

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

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

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

[0121] (5) After the primary antibody blocking was completed, the membrane was washed three times with PBS, and a fluorescently labeled secondary antibody (1:300 dilution) was added and incubated in the dark at room temperature for 1 h.

[0122] (6) After the secondary antibody incubation, the cells were washed three times with PBS, and DAPI was added to stain the cell nuclei. The cells were incubated in the dark at room temperature for 5 min.

[0123] (7) After nuclear staining, the slides were washed three times with PBS, sealed with anti-fluorescence quencher, and observed and photographed under a confocal microscope.

[0124] 3. Results

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

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

[0127] At different time points after PEDV infection and at different MOIs, cell RNA samples were collected and the mRNA expression level of the viral M gene was detected by q-PCR. Figure 3As shown, the results showed that after CLDN4 overexpression, the expression level of the viral M gene was significantly reduced at various time points of infection (12h, 24h and 36h) and under different MOI (0.01, 0.05 and 0.1MOI) infection conditions, that is, increased CLDN4 expression level can inhibit PEDV infection.

[0128] At different time points after PEDV infection and at different MOIs, cell protein samples and cell slide samples were collected, and the expression level of viral PEDV N protein was detected by Western blot and indirect immunofluorescence techniques. Figure 4 and Figure 5 As shown in the results, after CLDN4 overexpression, the expression level of PEDV N protein was significantly reduced under different infection times (12h, 24h and 36h) and different MOI (0.01, 0.05 and 0.1MOI) infection conditions, that is, increased CLDN4 expression level can inhibit PEDV infection.

[0129] 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 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 the prevention and control of porcine epidemic diarrhea virus infection.

2. Use 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 the prevention and control of porcine epidemic diarrhea virus infection.

3. Use of a recombinant vector comprising the porcine epidemic diarrhea resistance-associated 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 the prevention and control of 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 the prevention and control of 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. Use of a reagent for detecting the expression level of the porcine epidemic diarrhea resistance-related gene CLDN4 in the preparation of a detection kit for resistance to porcine epidemic diarrhea virus infection.

6. Use of the 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 pig's resistance to epidemic diarrhea virus infection is improved. The gene accession number of the CLDN4 in the NCBI database is XM_008018331.2.

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