Use of expression activator of fdxr gene in preparation of product for resisting porcine epidemic diarrhea virus infection

Abstract

The application belongs to the field of animal genetic engineering and molecular biotechnology, and particularly relates to FDXR The application of the expression activator of the gene in the preparation of a product for resisting porcine epidemic diarrhea virus infection. FDXR The nucleotide sequence of the gene is shown as SEQ ID NO. 1. The research result of the application shows that the expression level of the gene will affect the replication level of PEDV. FDXR When the expression of the gene in a host cell is inhibited, the replication of PEDV can be significantly promoted and the titer of the virus is increased. FDXR When the FDXR is overexpressed in the host cell, the replication of PEDV can be significantly inhibited and the titer of the virus is decreased. FDXR The experimental result not only shows that the gene can be used as a new target for the development of an antiviral drug for PEDV, but also provides a tool and material for the research on the molecular mechanism of the gene in the regulation of the replication of a pathogenic microorganism in a cell. FDXR The application belongs to the field of animal genetic engineering and molecular biotechnology, and particularly relates to

Description

Technical Field

[0001] This invention belongs to the fields of animal genetic engineering and molecular biology, specifically involving FDXR Application of gene expression activators in the preparation of products for treating porcine epidemic diarrhea virus infection. Background Technology

[0002] Porcine epidemic diarrhea (PED) is an intestinal infectious disease of pigs caused by porcine epidemic diarrhea virus (PEDV). Clinically, it mainly manifests as acute watery diarrhea, vomiting, and dehydration in piglets, while adult pigs often present with mild diarrhea or latent infection. This disease can lead to high mortality rates in piglets, causing significant economic losses to the pig farming industry. Although PEDV can proliferate in IPEC-J2 cells, its replication efficiency is low, severely hindering the progress of PEDV-related research and the development of control products. Currently, the main clinical product for PED control is the PED-TGE bivalent inactivated vaccine. This vaccine has drawbacks such as a short period of immune protection, the need for multiple immunizations, and a lack of specific interventions targeting key host cell targets of PEDV infection, making it difficult to achieve long-lasting and effective control. Summary of the Invention

[0003] To overcome the shortcomings of current PED prevention and control products, such as short-term immune protection, the need for multiple immunizations, and the lack of specific interventions targeting key host cell targets of PEDV infection, thus hindering the achievement of long-lasting and effective prevention and control, this invention provides... FDXR Application of gene expression activators in the preparation of products for treating porcine epidemic diarrhea virus infection.

[0004] To achieve the above objectives, the present invention adopts the following technical solution.

[0005] Ferredoxin reductase (FDXR), a key protein regulating mitochondrial ferredoxins, indirectly participates in three major physiological processes—steroidogenesis, heme synthesis, and Fe / S cluster biosynthesis—by transferring electrons to two human mitochondrial ferredoxins (FDX1 and FDX2) (see "Humans possess two mitochondrial ferredoxins, Fdx1 and Fdx2, with distinct roles insteroidogenesis, heme, and Fe / S cluster biosynthesis. Proc Natl Acad Sci U SA. 2010 Jun 29;107(26):11775-11780"). The gene encoding ferredoxin reductase is... FDXR Genes. Currently, FDXR Gene regulators are primarily used in research and product development for human-related diseases or other non-viral diseases (see "Hepatic ferredoxin reductase modulates mitochondrial function and ironhomeostasis in metabolic dysfunction-associated steatotic liver disease. ResSq [Preprint]. 2025 Aug 27:rs.3.rs-7014857"). Their application scope does not include the regulation of PEDV replication, based on... FDXR There is currently a lack of gene regulators targeting PEDV replication regulation. Given the shortcomings of current PED control products, such as poor efficacy and limited selection, there is an urgent need to expand the market. FDXR This invention aims to expand the application scope of gene regulators and explore their use in the preparation of products that regulate porcine epidemic diarrhea virus (PED) replication, thereby filling existing technological gaps and meeting the needs of the pig industry for efficient PED control. Based on this, the purpose of this invention is to provide... FDXR The application of gene expression activators in the preparation of products resistant to porcine epidemic diarrhea virus infection. FDXR The amino acid sequence of the gene is shown in SEQ ID NO.1.

[0006] This invention discovers FDXR Genes serve as key host targets for PEDV infection, providing FDXR A novel application of gene expression activators in the preparation of anti-PEDV drugs, which can directly act on the host-dependent process of viral replication by upregulating gene expression. FDXRInhibiting PEDV infection through gene expression breaks through the single-action mode of traditional vaccines that only induce immune responses, realizing a strategic shift from "passive immune prevention" to "active targeted intervention." This overcomes the shortcomings of existing vaccines, such as short immune protection period and the need for multiple immunizations, and provides a specific small molecule drug selection targeting key host cells for PED prevention and control, achieving a long-lasting and effective antiviral effect.

[0007] The present invention also provides the above. FDXR The use of gene expression activators in the preparation of products for treating porcine epidemic diarrhea virus infection, wherein the products are selected from any of the following: Cultures used to inhibit the replication of swine epidemic diarrhea virus and raw materials for vaccine production.

[0008] Drugs used to treat swine epidemic diarrhea virus infection or replication.

[0009] A cell model resistant to swine epidemic diarrhea virus infection.

[0010] Preferably, the cell model for resisting porcine epidemic diarrhea virus infection is obtained through the... FDXR The cells were constructed by treating porcine cells with gene expression activators.

[0011] Preferably, the FDXR Gene expression activators include FDXR Gene overexpression vectors, CRISPRa activation systems, or transcription activators.

[0012] Preferably, the FDXR Gene overexpression vectors include pcDNA3.1-FDXR.

[0013] The present invention also provides the above. FDXR The use of gene expression inhibitors in the preparation of products that promote porcine epidemic diarrhea virus infection, wherein the products are selected from any of the following: Cultures used to promote the replication of porcine epidemic diarrhea virus and raw materials for vaccine production.

[0014] Drugs that promote infection or replication of porcine epidemic diarrhea virus.

[0015] A cell model that promotes porcine epidemic diarrhea virus infection.

[0016] Preferably, the cell model promoting porcine epidemic diarrhea virus infection is obtained through the... FDXR The cells were constructed by treating porcine cells with gene expression inhibitors.

[0017] Preferably, the FDXRThe gene expression inhibitor is selected from any one or more of shRNA, siRNA, miRNA, antisense oligonucleotides, gene editing tools, and small chemical molecule inhibitors.

[0018] Preferably, the nucleotide sequence of the shRNA is shown in SEQ ID NO.2.

[0019] Preferably, the gene editing tool is a CRISPR-Cas9 system, and the CRISPR-Cas9 system includes a targeting mechanism. FDXR The sgRNA of a gene.

[0020] Preferably, the application includes: placing the FDXR Gene expression inhibitors act on porcine cells to obtain... FDXR Gene knockdown cells were used for high-titer culture of porcine epidemic diarrhea virus.

[0021] This invention also provides a method for preparing high-titer porcine epidemic diarrhea virus, comprising the following steps: Using the FDXR Treating porcine cells with gene expression inhibitors yields... FDXR Gene knockdown cells.

[0022] Porcine epidemic diarrhea virus infection FDXR Gene knockdown cells were then cultured.

[0023] High-titer porcine epidemic diarrhea virus was obtained by separating the cell culture supernatant.

[0024] Preferably, the FDXR The gene expression inhibitor is shRNA with the sequence shown in SEQ ID NO.2.

[0025] Preferably, the porcine-derived cells are porcine small intestinal epithelial cells (e.g., IPEC-J2).

[0026] The present invention also provides a method for knockdown pigs FDXR Interfering RNA for genes, including shRNA with a sequence as shown in SEQ ID NO.2.

[0027] The present invention also provides for FDXR The use of gene / FDXR protein-targeting activators in the preparation of drugs (e.g., antiviral drugs) for the prevention and / or treatment of porcine epidemic diarrhea virus infection.

[0028] Preferably, the drug is a preparation or reagent for promoting the expression of the porcine FDXR gene.

[0029] Preferably, the pig FDXRThe gene was selected from the FDXR gene with NCBI accession number NM_001244727.1, or the FDXR protein coding sequence shown in SEQ.ID.NO.1.

[0030] Preferably, the formulation comprises a porcine FDXR protein overexpression vector (e.g., pcDNA3.1-FDXR), which, upon introduction (e.g., transfection) into the aforementioned porcine cells, activates the cells by increasing the expression level of FDXR protein in the corresponding cells. FDXR Genes (i.e., those that activate the expression of the FDXR protein) are used to suppress the replication of porcine epidemic diarrhea virus.

[0031] Preferably, the reagent comprises porcine... FDXR Drug molecules that target genes and can increase the expression level of FDXR protein.

[0032] The present invention also provides a cell model for resisting porcine epidemic diarrhea virus infection, which is generated by using a method to promote the growth of pigs... FDXR It is made by treating the above-mentioned porcine cells with gene expression agents or reagents.

[0033] Preferably, the formulation comprises a porcine FDXR protein overexpression vector (e.g., pcDNA3.1-FDXR).

[0034] Preferably, the reagent comprises porcine... FDXR Drug molecules that target genes and can increase the expression level of FDXR protein.

[0035] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention provides FDXR The application of gene expression activators in the preparation of products against porcine epidemic diarrhea virus (PEDV) infection provides a specific intervention method targeting key host cell targets of PEDV infection, expanding the application of gene expression activators in the production of products against PEDV infection. FDXR The application scope of genes in the prevention and control of viral diseases in livestock and poultry. This invention discovers... FDXR Genes serve as key host targets for PEDV infection, providing FDXR A novel application of gene expression activators in the preparation of anti-PEDV drugs, which can directly act on the host-dependent process of viral replication by upregulating gene expression. FDXR Inhibiting PEDV infection through gene expression breaks through the single-action mode of traditional vaccines that only induce immune responses, realizing a strategic shift from "passive immune prevention" to "active targeted intervention." This overcomes the shortcomings of existing vaccines, such as short immune protection period and the need for multiple immunizations, and provides a specific small molecule drug selection targeting key host cells for PED prevention and control, achieving a long-lasting and effective antiviral effect.

[0036] 2. This invention, through the construction of an FDXR overexpression vector, is the first to discover that increasing the expression level of FDXR in porcine cells (such as IPEC-J2 cells) can reduce the replication level of porcine epidemic diarrhea virus. This finding suggests that... FDXR Activators of the gene (or FDXR protein) can be used in the development of novel antiviral drugs targeting porcine epidemic diarrhea virus infection. Additionally, this invention utilizes RNA interference technology to knock down porcine cells. FDXR Gene, first discovery of inhibition FDXR The expression of this gene can promote the replication of porcine epidemic diarrhea virus within cells and increase the viral titer in cell culture supernatants. This finding suggests that... FDXR Gene inhibitors can be used to obtain high titers of porcine epidemic diarrhea virus.

[0037] This invention is based on the changes in FDXR expression levels in porcine epidemic diarrhea virus-infected porcine cells (such as IPEC-J2 cells), and knocks down FDXR in the corresponding host cells. FDXR Experiments on gene and FDXR overexpression not only rely on experimental results FDXR Genetic identification revealed it to be a resistance gene against porcine epidemic diarrhea virus (i.e., promoting immunity). FDXR Gene expression inhibits the replication of porcine epidemic diarrhea virus, and when FDXR When gene expression was suppressed, the replication level of porcine epidemic diarrhea virus (PEDV) was significantly increased, and the viral load was also elevated. Furthermore, it was found that the virus could effectively suppress [the virus]. FDXR Interfering RNA for gene expression (also acquired for promoting) FDXR FDXR overexpression plasmid for gene expression, and for research FDXR This study provides important tools and materials (including highly efficient viral amplification cells and high-titer viruses) for understanding the molecular mechanisms by which genes regulate the replication of pathogenic microorganisms within cells and for the preparation of porcine epidemic diarrhea virus vaccines. Attached Figure Description

[0038] Figure 1 The results show the FDXR expression level after PEDV infection of IPEC-J2 in this invention; wherein: A represents the expression level of FDXR protein in IPEC-J2 cells at 8 h and 16 h post-PEDV infection, detected by Western blot (β-actin was used as an internal control). B represents the intracellular contents of IPEC-J2 cells detected by real-time quantitative PCR at 8 h and 16 h post-PEDV infection. FDXR Gene mRNA level; **** indicates that, relative to the PEDV-uninfected group (i.e., the MOCK group), FDXR Gene expression level p <0.0001).

[0039] Figure 2 The results of the construction of FDXR overexpression vectors and the identification of overexpression effects in this invention are as follows: A is FDXR Identification of PCR products of the gene; lane M is DL 5000 plus DNA Marker, lane "negative" is the template-free negative control, lanes 1, 2, and 3 are all... FDXR PCR products of genes; Lane B is for pcDNA3.1-FDXR plasmid digestion identification; lane M is for DL ​​5000 plus DNA Marker, lane "Plasmid" is the undigested plasmid, and lane "Double Digestion" is the recombinant plasmid EcoRI / SalI double digestion. C represents the expression level of FDXR protein in IPEC-J2 cells detected by Western blot; β-actin is the internal control; pcDNA3.1 group is the negative control of the empty vector in the overexpression group; and pcDNA3.1-FDXR group, i.e., the overexpression group, is the cell transfected with pcDNA3.1-FDXR plasmid.

[0040] Figure 3 The results show the effect of FDXR overexpression on PEDV replication in this invention; wherein: A represents the detection of the porcine epidemic diarrhea virus N gene in IPEC-J2 cells by real-time quantitative PCR. PEDV N (Gene) mRNA level (PEDV group is cells infected with PEDV only, pcDNA3.1+PEDV group is cells transfected with empty vector and then infected with porcine epidemic diarrhea virus, pcDNA3.1-FDXR+PEDV group is cells transfected with constructed overexpression vector and then infected with porcine epidemic diarrhea virus, GAPDH is internal control). **** indicates relative to the PEDV group PEDV N Gene expression level p <0.0001, ns indicates relative to the PEDV group PEDV N Gene expression level p >0.05); B represents the detection of PEDV replication level (PEDV N protein expression level) and FDXR protein expression level in IPEC-J2 cells by Western blot (β-actin is used as an internal control). C represents the median tissue culture infection dose (TCID) of the virus in the supernatant of IPEC-J2 cell culture. 50The PEDV group consists of cells infected with PEDV only; the pcDNA3.1+PEDV group consists of cells transfected with an empty vector and then infected with PEDV; and the pcDNA3.1-FDXR+PEDV group consists of cells transfected with a constructed overexpression vector and then infected with PEDV. **** indicates the viral TCID relative to the PEDV group. 50 level p <0.0001, ns represents the viral TCID relative to the PEDV group. 50 level p >0.05.

[0041] Figure 4 In this invention FDXR Results of gene interference RNA identification: Western blot was used to detect the expression level of FDXR protein in IPEC-J2 cells after transfection with FDXR gene interference RNA; the shNC group was cells transfected with negative control shRNA, the shFDXR group was cells transfected with FDXR gene shRNA, and β-actin was used as an internal control.

[0042] Figure 5 Interference in this invention FDXR Results of the effect of gene expression on PEDV replication; among which: A represents the detection of the porcine epidemic diarrhea virus N gene in IPEC-J2 cells by real-time quantitative PCR. PEDV N (Gene) mRNA level (PEDV group: cells infected with PEDV only; shNC+PEDV group: cells transfected with negative control shRNA and then infected with PEDV; shFDXR+PEDV group: cells transfected with...) FDXR Cells were then infected with PEDV after receiving shRNA of the gene, with GAPDH as an internal control; *** indicates the expression level of the PEDV N gene relative to the PEDV group. p <0.001, ns represents the PEDV N gene expression level relative to the PEDV group. p >0.05); B represents the detection of PEDV replication level (PEDV N protein expression level) and FDXR protein expression level in IPEC-J2 cells by Western blot (β-actin is used as an internal control). C represents the median tissue culture infection dose (TCID) of the virus in the supernatant of IPEC-J2 cell culture. 50 The PEDV group consisted of cells infected with PEDV only; the shNC+PEDV group consisted of cells transfected with negative control shRNA and then infected with PEDV; the shFDXR+PEDV group consisted of cells transfected with shRNA containing the FDXR gene and then infected with PEDV; **** indicates the viral TCID relative to the PEDV group.50 level p <0.0001, ns represents the viral TCID relative to the PEDV group. 50 level p >0.05. Detailed Implementation

[0043] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0044] Example 1 1. FDXR expression level after PEC-J2 inoculation Materials and reagents: IPEC-J2 cells were preserved by the Animal Major Disease Novel Vaccine R&D Innovation Team of the College of Veterinary Medicine, Northwest A&F University (refer to "PoRVA G9P

[23] and G5P[7] infections differentially promote PEDV replication by reprogramming glutamine metabolism. PLoS Pathog. 2024 Jun 21;20(6):e1012305"); Primary antibody: FDXR antibody (rabbit source) was purchased from Wuhan Sanying Biotechnology Co., Ltd., PEDV The N protein antibody (rabbit-derived) was prepared by the Animal Major Disease Novel Vaccine R&D Innovation Team of the College of Veterinary Medicine, Northwest A&F University (refer to "Isolation and Pathogenicity Analysis of a GIIa Type Porcine Epidemic Diarrhea Virus [J]. Journal of Animal Husbandry and Veterinary Medicine, 2025, 56(08):4101-4111"); the β-actin antibody (rabbit-derived) was purchased from Wuhan Sanying Biotechnology Co., Ltd.; the PEDVLZ202401 strain was preserved ... Isolation and pathogenicity analysis of viruses [J]. Journal of Animal Husbandry and Veterinary Medicine, 2025, 56(08):4101-4111”); Secondary antibody: Horseradish peroxide (HRP) labeled goat anti-rabbit IgG antibody was purchased from Wuhan Sanying Biotechnology Co., Ltd.; mRNA reverse transcription kit was purchased from Nanjing Novizan Biotechnology Co., Ltd.; qPCR kit was purchased from Wuhan Aibote Biotechnology Co., Ltd.; PVDF membrane was purchased from Roche Pharmaceuticals Ltd.; ECL chromogenic solution was purchased from Aoruisaisi Biotechnology (Chengdu) Co., Ltd.; other routine reagents were all analytical grade.

[0045] After IPEC-J2 cells were infected with PEDV LZ202401 (MOI=1), cells were collected at 8 h and 16 h (specifically, the culture medium was removed, and the cells were washed). Cells were lysed with RIPA lysis buffer containing a protease inhibitor, lysed on ice for 30 min, then loaded with loading buffer and boiled for 10 min. SDS-PAGE was then performed, followed by transfer to a PVDF membrane. After blocking with 5% (w / v) skim milk powder at room temperature for 2 h, the membrane was treated with primary and secondary antibodies before imaging. Viral replication (PEDV N protein expression) and FDXR protein expression are shown below. Figure 1 As shown in A, the results indicate that PEDV LZ202401 infection leads to increased FDXR protein expression in its host cells (IPEC-J2 cells).

[0046] After IPEC-J2 cells were infected with PEDV LZ202401 with MOI=1, cells were collected at 8h and 16h, respectively. Total RNA was extracted from the collected cells using the TRIZOL precipitation method. The extracted total RNA was then reverse transcribed into cDNA. Specifically, the reverse transcription reaction system (Table 1) was placed in a PCR instrument and incubated at 37℃ for 15min. After incubation, the reverse transcriptase was inactivated by heating at 85℃ for 5s.

[0047] Table 1. Reverse transcription system for cDNA synthesis Using cDNA obtained from reverse transcription as a template, intracellular cDNA was detected using qPCR primers (Table 3). FDXR At the gene mRNA level, specifically, the qPCR reaction system (Table 2) was placed in a PCR instrument and pre-denatured at 95℃ for 20s, followed by 40 cycles of (95℃ for 15s, 60℃ for 20s).

[0048] Table 2 qPCR reaction system Table 3 qPCR primers Real-time quantitative PCR results showed that PEDV LZ202401 infection significantly reduced the FDXR gene mRNA level in its host cells IPEC-J2. Figure 1 (B in the middle).

[0049] 2. Construction of FDXR overexpression vector Materials and reagents: IPEC-J2 cells were preserved by the Animal Major Disease Novel Vaccine R&D Innovation Team of the College of Veterinary Medicine, Northwest A&F University (refer to "PoRVA G9P

[23] and G5P[7] infections differentially promote PEDV replication by reprogramming glutamine metabolism. PLoS Pathog. 2024 Jun 21;20(6):e1012305"); plasmid extraction kits were purchased from Tiangen Biotech (Beijing) Co., Ltd. EcoR I enzyme, Sale Enzyme I was purchased from Jiangsu Bristol-Myers Squibb Biotechnology Co., Ltd.; Phanta Max high-fidelity enzyme was purchased from Nanjing Novizan Biotechnology Co., Ltd.; pcDNA3.1 vector (trade name pcDNA3.1(+)) was purchased from Beijing Qingke Biotechnology Co., Ltd.; mRNA reverse transcription kit was purchased from Nanjing Novizan Biotechnology Co., Ltd.; all other routine reagents were of analytical grade.

[0050] According to data published by the NCBI, pigs FDXR Gene (abbreviated as) FDXR The gene (accession number NM_001244727.1, nucleotide sequence shown in SEQ ID NO.1) was designed to amplify... FDXR Primers for the gene coding sequence (Table 4) were synthesized by Beijing Qingke Biotechnology Co., Ltd., and their sequences are as follows: The nucleotide sequence of the upstream primer FDXR-F is shown in SEQ.ID.NO.8: 5'-CGGAATTCATGGCTTCGCGCTGCTGGCG-3'.

[0051] The nucleotide sequence of the downstream primer FDXR-R is shown in SEQ.ID.NO.9: 5'-GCGTCGACTCAGTGGCCCAGGAGGCGC-3'.

[0052] Table 4 FDXR Gene Total RNA was extracted from uninfected PEDV-positive IPEC-J2 cells using the TRIZOL precipitation method, and then reverse transcribed into cDNA (reverse transcription reaction system is shown in Table 1). The target gene was then amplified by PCR using the cDNA as a template, and the PCR products were subjected to agarose gel electrophoresis. The amplification system is shown in Table 5, and the reaction procedure is shown in Table 6. The agarose gel containing the PCR product was excised under UV light, and the PCR product containing the target gene was recovered using a DNA purification and recovery kit. Figure 2 (A in the middle).

[0053] Table 5 PCR reaction system Table 6 PCR Reaction Procedure Using restriction endonucleases EcoR I and Sale I. The PCR product and pcDNA3.1 vector were double-digested with enzymes, as shown in Table 7. The reaction conditions were: 37℃ water bath for 2 h. Then, the digested products were electrophoresed on a 1% agarose gel. The agarose gel containing the target gene and vector backbone was excised under UV light and recovered using a DNA purification and recovery kit.

[0054] Table 7 Enzyme digestion system The ligation system was prepared using the recovered target gene and vector backbone (Table 8), and then the ligation system was placed in a 16℃ metal bath for 10 h for ligation.

[0055] Table 8 Connection System Competent *E. coli* were placed on ice and thawed. All ligation products were added, and the mixture was gently pipetted to mix. The mixture was then incubated on ice for 30 min. Afterward, the cells were heat-activated in a 42°C water bath for 30 s, quickly transferred to an ice bath, and incubated for 5 min. 900 μL of antibiotic-free LB broth was added, and the mixture was incubated at 37°C on a shaker at 200 rpm for 1 h. The cells were collected by centrifugation at 2500 rpm for 5 min, and the precipitate was resuspended in 100 μL of antibiotic-free LB broth. The precipitate was then evenly spread onto LB solid medium containing ampicillin and incubated upside down at 37°C for 10 h. Single colonies were picked using an inoculation loop for culture and PCR identification. Positive bacteria were then expanded using LB broth containing ampicillin, and plasmids were extracted for restriction enzyme digestion identification. Specifically, recombinant plasmids were extracted using an endotoxin-free plasmid extraction kit, and then digested with restriction endonucleases. EcoR I and Sale I underwent double digestion (the digestion system is shown in Table 7), and the results are as follows: Figure 2As shown in B in the diagram. The positive bacterial culture was sent to Beijing Qingke Biotechnology Co., Ltd. for recombinant plasmid sequencing. After sequence alignment, the eukaryotic expression plasmid pcDNA3.1-FDXR was obtained. The corresponding positive bacteria were preserved at -80℃ using the glycerol preservation method. The eukaryotic expression plasmid pcDNA3.1-FDXR is also known as the pcDNA3.1-FDXR plasmid.

[0056] 3. Verify the intracellular expression of the FDXR overexpression vector. Materials and reagents: IPEC-J2 cells were preserved by the Animal Major Disease Novel Vaccine R&D Innovation Team of the College of Veterinary Medicine, Northwest A&F University (refer to "PoRVA G9P

[23] and G5P[7] infections differentially promote PEDV replication by reprogramming glutamine metabolism. PLoS Pathog.2024 Jun 21;20(6):e1012305"); Primary antibody: FDXR antibody (rabbit source) was purchased from Wuhan Sanying Biotechnology Co., Ltd., β-actin antibody (rabbit source) was purchased from Wuhan Sanying Biotechnology Co., Ltd.; Secondary antibody: horseradish peroxide (HRP) labeled goat anti-rabbit IgG antibody was purchased from Wuhan Sanying Biotechnology Co., Ltd.; PVDF membrane was purchased from Roche Pharmaceuticals Ltd., Shanghai; ECL chromogenic solution was purchased from Aorui Sais Biotechnology (Chengdu) Co., Ltd.; Lipo6000 was purchased from Beyotime Biotechnology Co., Ltd., Shanghai; other conventional reagents were all analytical grade.

[0057] IPEC-J2 cells were cultured to 80% confluency, then digested with trypsin, mixed with DMEM / F12 medium containing 10% (w / v) FBS, and seeded into 12-well plates. Once the cell density reached 80%, plasmid transfection was performed. Before transfection, the old culture medium was discarded, the cells were washed once with PBS, and 500 μL of DMEM / F12 medium was added. In a 1.5 mL RNase-free centrifuge tube, pcDNA3.1-FDXR plasmid, transfection reagent (plasmid to Lipo6000 transfection reagent ratio of 1:2 (μg / μL), plasmid dosage 1 μg / well), and 100 μL of DMEM / F12 medium were added, mixed thoroughly, and allowed to stand for 25 min to prepare the transfection complex. This complex was then added dropwise to the culture wells. The pcDNA3.1 vector was processed in the same manner. The culture plates were incubated at 37°C in a 5% CO2 incubator for 24 h.

[0058] Cells were collected after 24 hours of culture and lysed with RIPA lysis buffer containing 1 mM protease inhibitor (PMSF). After lysis on ice for 30 minutes, loading buffer was added and the cells were boiled for 10 minutes. SDS-PAGE was then performed, followed by transfer to a PVDF membrane. The membrane was blocked with 5% (w / v) skim milk at room temperature for 2 hours, then coated with primary and secondary antibodies before development. FDXR protein expression was shown below. Figure 2 As shown in C, the results indicate that the pcDNA3.1-FDXR plasmid achieved overexpression of the FDXR protein in the host cells of porcine epidemic diarrhea virus (IPEC-J2 cells).

[0059] 4. Effect of expressing the porcine FDXR gene on PEDV replication Materials and reagents: IPEC-J2 cells and Vero cells were preserved by the Animal Major Disease Novel Vaccine R&D Innovation Team of the College of Veterinary Medicine, Northwest A&F University (refer to "PoRVA G9P

[23] and G5P[7] infections differentially promote PEDV replication by reprogramming glutamine metabolism. PLoS Pathog. 2024 Jun 21;20(6):e1012305"); PEDV LZ202401 strain was preserved by the Animal Major Disease Novel Vaccine R&D Innovation Team of the College of Veterinary Medicine, Northwest A&F University (refer to "Isolation and pathogenicity analysis of a strain of GⅡa porcine epidemic diarrhea virus[J]. Journal of Animal Husbandry and Veterinary Medicine, 2025, 56(08):4101-4111"); Lipo6000 was purchased from Shanghai Beyotime Biotechnology Co., Ltd.; Primary antibody: FDXR antibody (rabbit source) was purchased from Wuhan Sanying Biotechnology Co., Ltd., PEDV The N protein antibody (rabbit-derived) was prepared by the Animal Major Disease Novel Vaccine R&D Innovation Team of the College of Veterinary Medicine, Northwest A&F University (refer to "Isolation and Pathogenicity Analysis of a GIIa Type Porcine Epidemic Diarrhea Virus [J]. Journal of Animal Husbandry and Veterinary Medicine, 2025, 56(08):4101-4111"). The β-actin antibody (rabbit-derived) was purchased from Wuhan Sanying Biotechnology Co., Ltd.; the secondary antibody: horseradish peroxide (HRP) labeled goat anti-rabbit IgG antibody was purchased from Wuhan Sanying Biotechnology Co., Ltd.; the mRNA reverse transcription kit was purchased from Nanjing Novizan Biotechnology Co., Ltd.; the qPCR kit was purchased from Wuhan Aibotek Biotechnology Co., Ltd.; the PVDF membrane was purchased from Roche Pharmaceuticals Ltd. in Shanghai; the ECL chromogenic solution was purchased from Aoruisaisi Biotechnology (Chengdu) Co., Ltd.; and all other routine reagents were of analytical grade.

[0060] IPEC-J2 cells were cultured to 80% density, digested with trypsin, and resuspended in DMEM / F12 medium containing 10% (w / v) FBS. Cell counts were then performed. 1 μg of pcDNA3.1-FDXR plasmid was added to 1×10⁻⁶ cells. 5 Cells were transfected at a ratio of 100 μL per cell in culture plates using liposomes (i.e., Lipo6000 transfection reagent). Cell groups were prepared by transfecting empty vector (pcDNA3.1 vector), virus-infected cells (without plasmid transfection), and normal cells (without plasmid transfection and virus infection). After transfection, the culture plates were incubated at 37°C in a 5% CO2 incubator for 12 h, then infected with PEDV LZ202401 at MOI=1. After 16 h, porcine epidemic diarrhea virus replication (mRNA and protein expression) levels were measured, and viral TCID in the cell culture supernatant was also detected. 50 .

[0061] mRNA levels: Cells were collected 16 h after infection with PEDV LZ202401. Total RNA was extracted from the collected cells using the TRIZOL precipitation method. The extracted total RNA was then reverse transcribed into cDNA. Specifically, the reverse transcription reaction system (refer to Table 1) was incubated in a PCR instrument at 37°C for 15 min, followed by heating at 85°C for 5 s to inactivate the reverse transcriptase. The cDNA obtained from reverse transcription was used as a template, and intracellular mRNA was detected using qPCR primers (Table 9). PEDV N The mRNA level was specifically determined by placing the qPCR reaction system (refer to Table 2) in a PCR instrument for 95℃ pre-denaturation for 20s followed by 40 cycles of (95℃ 15s, 60℃ 20s). Real-time quantitative PCR results showed that the PEDV N gene mRNA level was suppressed in cells transfected with pcDNA3.1-FDXR plasmid 16 hours after porcine epidemic diarrhea virus (PEDV) infection. Figure 3 (A in the original text). This result suggests that the intracellular porcine epidemic diarrhea virus replication level decreases with increased FDXR gene expression.

[0062] Table 9. qPCR primers Protein expression levels: Cells were collected 16 hours after infection with porcine epidemic diarrhea virus (PEDV). Cells were lysed with RIPA lysis buffer containing 1 mM protease inhibitor (PMSF), lysed on ice for 30 min, then loaded with loading buffer and boiled for 10 min. SDS-PAGE was then performed, followed by transfer to a PVDF membrane. After blocking with 5% (w / v) skim milk powder at room temperature for 2 h, the membrane was treated with primary and secondary antibodies before development. The expression levels of FDXR and PEDV N proteins in the cells are shown below. Figure 3As shown in B, the expression level of porcine epidemic diarrhea virus protein decreased after overexpression of FDXR.

[0063] TCID 50 Experiment: Cells transfected with pcDNA3.1 vector, pcDNA3.1-FDXR plasmid, and untransfected cells were collected and supernatant was collected 16 hours after viral infection. Vero cells in logarithmic growth phase were resuspended one day in advance and the cell concentration was adjusted to 8 × 10⁶ cells / year. 4 Cells / mL were evenly inoculated into 96-well plates, with 8 replicates for each viral concentration gradient. The cell culture medium was discarded when the cell density reached approximately 80%. The cell culture supernatant collected after inoculation was serially diluted with DMEM medium containing 10 μg / mL trypsin, with a dilution factor of 10-10. -1 Up to 10 -10 Cell culture supernatant was added at 100 μL per well to the above 96-well plate, while an equal volume of DMEM medium containing 10 μg / mL trypsin was added to the blank control wells. After waiting for virus adsorption for 2 hours, the cell culture medium was replaced with DMEM medium containing 10 μg / mL trypsin. CPE was observed daily. Once the cell condition in each well stabilized and the number of diseased wells no longer increased, the viral titer (TCID) of the amplified porcine epidemic diarrhea virus was calculated using the Reed-Muench method. 50 ), through TCID 50 The results of detecting viral titers in cell culture supernatants are as follows: Figure 3 As shown in C, compared with the PEDV-infected group, overexpression of FDXR followed by PEDV infection can significantly reduce the viral titer of PEDV in the cell culture supernatant.

[0064] 5. Evaluation of the effectiveness of interfering RNA in the porcine FDXR gene Materials and reagents: IPEC-J2 cells were preserved by the Animal Major Disease Novel Vaccine R&D Innovation Team of the College of Veterinary Medicine, Northwest A&F University (refer to "PoRVA G9P

[23] and G5P[7] infections differentially promote PEDV replication by reprogramming glutamine metabolism. PLoS Pathog.2024 Jun 21;20(6):e1012305"); Primary antibody: FDXR antibody (rabbit source) was purchased from Wuhan Sanying Biotechnology Co., Ltd., β-actin antibody (rabbit source) was purchased from Wuhan Sanying Biotechnology Co., Ltd.; Secondary antibody: horseradish peroxide (HRP) labeled goat anti-rabbit IgG antibody was purchased from Wuhan Sanying Biotechnology Co., Ltd.; PVDF membrane was purchased from Roche Pharmaceuticals Ltd., Shanghai; ECL chromogenic solution was purchased from Aorui Sais Biotechnology (Chengdu) Co., Ltd.; Lipo6000 was purchased from Beyotime Biotechnology Co., Ltd., Shanghai; other conventional reagents were all analytical grade.

[0065] According to data published by the NCBI, pigs FDXR Gene (NM_001244727.1) was used to design an shRNA (shFDXR) that could interfere with the FDXR gene encoding (Table 4). Beijing Qingke Biotechnology Co., Ltd. synthesized the shFDXR into the pSIH1-H1-copGFP-T2A-Puro vector for experimental use. The shFDXR sequence is as follows: The nucleotide sequence of shFDXR sense is shown in SEQ.ID.NO.2: 5'-GATCCGCTGAGCAGCATAGGGTATAACTCGAGTTATACCCTATGCTGCTCAGCTTTTTG-3'.

[0066] The nucleotide sequence of shFDXR anti-sense is shown in SEQ.ID.NO.3: 5'-AATTCAAAAAGCTGAGCAGCATAGGGTATAACTCGAGTTATACCCTATGCTGCTCAGCG-3'.

[0067] The negative control shRNA used in the experiment was the pSIH1-H1-copGFP-T2A-Puro empty vector (shNC for short).

[0068] The cultured IPEC-J2 cells were digested and counted beforehand, and then diluted to 2×10⁶ cells / mL with DMEM / F12 medium containing 10% (w / v) FBS. 41 mL of cell suspension at a concentration of 1 cell / mL was added to each well of the culture plate. Transfection was performed when the cell density reached 80%. Negative control shRNA and shFDXR were transfected into IPEC-J2 cells using Lipo6000 transfection reagent (shRNA to Lipo6000 transfection reagent concentration: 1:2). After transfection, the culture plate was placed in a 37°C, 5% CO2 incubator. Cells were collected after 16 h and lysed with RIPA lysis buffer containing 1 mM protease inhibitor (PMSF). After lysis on ice for 30 min, loading buffer was added and the cells were boiled for 10 min. SDS-PAGE was then performed, and the cells were transferred to a PVDF membrane. After blocking with 5% skim milk at room temperature for 2 h, the membrane was treated with primary and secondary antibodies before development. Western blot analysis showed that shFDXR transfected into the cells had a high interference efficiency against the FDXR gene. Figure 4 ).

[0069] 6. Effects of interfering with porcine FDXR gene expression on porcine epidemic diarrhea virus replication Materials and reagents: IPEC-J2 cells and Vero cells were preserved by the Animal Major Disease Novel Vaccine R&D Innovation Team of the College of Veterinary Medicine, Northwest A&F University (refer to "PoRVA G9P

[23] and G5P[7] infections differentially promote PEDV replication by reprogramming glutamine metabolism. PLoS Pathog. 2024 Jun 21;20(6):e1012305"); PEDV LZ202401 strain was preserved by the Animal Major Disease Novel Vaccine R&D Innovation Team of the College of Veterinary Medicine, Northwest A&F University (refer to "Isolation and pathogenicity analysis of a strain of GⅡa porcine epidemic diarrhea virus[J]. Journal of Animal Husbandry and Veterinary Medicine, 2025, 56(08):4101-4111"); Lipo6000 was purchased from Shanghai Beyotime Biotechnology Co., Ltd.; Primary antibody: FDXR antibody (rabbit source) was purchased from Wuhan Sanying Biotechnology Co., Ltd., PEDV The N protein antibody (rabbit-derived) was prepared by the Animal Major Disease Novel Vaccine R&D Innovation Team of the College of Veterinary Medicine, Northwest A&F University (refer to "Isolation and Pathogenicity Analysis of a GIIa Type Porcine Epidemic Diarrhea Virus [J]. Journal of Animal Husbandry and Veterinary Medicine, 2025, 56(08):4101-4111"). The β-actin antibody (rabbit-derived) was purchased from Wuhan Sanying Biotechnology Co., Ltd.; the secondary antibody: horseradish peroxide (HRP) labeled goat anti-rabbit IgG antibody was purchased from Wuhan Sanying Biotechnology Co., Ltd.; the mRNA reverse transcription kit was purchased from Nanjing Novizan Biotechnology Co., Ltd.; the qPCR kit was purchased from Wuhan Aibotek Biotechnology Co., Ltd.; the PVDF membrane was purchased from Roche Pharmaceuticals Ltd. in Shanghai; the ECL chromogenic solution was purchased from Aoruisaisi Biotechnology (Chengdu) Co., Ltd.; and all other routine reagents were of analytical grade.

[0070] The cultured IPEC-J2 cells were digested and counted beforehand, and then diluted to 2×10⁶ cells / mL with DMEM / F12 medium containing 10% (w / v) FBS. 4 Add 1 mL of cell suspension to each well of the culture plate to a concentration of 1 cell / mL. Transfection can be performed when the cell density reaches 80%. Transfect the cells using Lipo6000 transfection reagent. FDXRThe gene shRNA, shFDXR (shFDXR+PEDV group), was used as the transfection site. Simultaneously, a negative control shRNA transfection group (shNC+PEDV group) and a PEDV group infected only with the virus but without shRNA transfection were established. After transfection, the culture plates were incubated at 37°C in a 5% CO2 incubator for 12 hours, and then infected with PEDV LZ202401 with MOI=1. After 16 hours, porcine epidemic diarrhea virus replication (mRNA and protein expression) levels were measured, and viral TCID in the cell culture supernatant was also detected. 50 .

[0071] mRNA level: Cells were collected 16 h after infection with PEDV LZ202401. Total RNA was extracted from the collected cells using the TRIZOL precipitation method, and then the extracted total RNA was reverse transcribed into cDNA. The intracellular PEDV N gene mRNA level was detected using the reverse-transcribed cDNA as a template and qPCR primers (Table 9). Real-time quantitative PCR results are shown below. Figure 5 As shown in Figure A, cells transfected with shFDXR can be seen 16 hours after infection with porcine epidemic diarrhea virus. PEDV N The expression level of the FDXR gene mRNA increased. This result indicates that the intracellular replication level of porcine epidemic diarrhea virus increases with the knockdown of the FDXR gene.

[0072] Protein expression levels: Cells were collected 16 hours after infection with porcine epidemic diarrhea virus (PEDV). Cells were lysed with RIPA lysis buffer containing 1 mM protease inhibitor (PMSF), lysed on ice for 30 min, then loaded with loading buffer and boiled for 10 min. SDS-PAGE was then performed, followed by transfer to a PVDF membrane. After blocking with 5% (w / v) skim milk powder at room temperature for 2 h, the membrane was treated with primary and secondary antibodies before development. The expression levels of FDXR and PEDV N proteins in the cells are shown below. Figure 5 As shown in B, knockdown can be observed. FDXR Genetic modification increases the expression of porcine epidemic diarrhea virus protein.

[0073] TCID 50 Experiment: Cells transfected with shNC, shFDXR, and untransfected shRNA were collected and their supernatants were collected 16 hours after inoculation. One day in advance, Vero cells in logarithmic growth phase were resuspended and their concentration adjusted to 8 × 10⁶ cells / cells. 4 Cells / mL were evenly inoculated into 96-well plates, with 8 replicates for each viral concentration gradient. The cell culture medium was discarded when the cell density reached approximately 80%. The cell culture supernatant collected after inoculation was serially diluted with DMEM medium containing 10 μg / mL trypsin, with a dilution factor of 10-10. -1 Up to 10 -10Cell culture supernatant was added at 100 μL per well to the above 96-well plate, while an equal volume of DMEM medium containing 10 μg / mL trypsin was added to the blank control wells. After waiting for virus adsorption for 2 hours, the cell culture medium was replaced with DMEM medium containing 10 μg / mL trypsin. CPE was observed daily. Once the cell condition in each well stabilized and the number of diseased wells no longer increased, the viral titer (TCID) of the amplified porcine epidemic diarrhea virus was calculated using the Reed-Muench method. 50 ), through TCID 50 The results of detecting viral titers in cell culture supernatants are as follows: Figure 5 As shown in C, compared to the PEDV-infected group, knockdown... FDXR Re-infection with PEDV after gene sequencing significantly increases the viral titer of PEDV in cell culture supernatant.

[0074] In summary, this invention has found through experiments that: (1) FDXR expression levels in host cells (e.g., IPEC-J2) increase after infection with porcine epidemic diarrhea virus; and (2) RNA interference technology can be used to inhibit host cells (e.g., IPEC-J2). FDXR After gene expression, the downregulation of FDXR expression promotes the replication of porcine epidemic diarrhea virus, resulting in a significant increase in both the in vitro replication level and viral titer of porcine epidemic diarrhea virus; (3) while the overexpressing pigs were introduced into host cells (e.g., IPEC-J2) FDXR Following plasmidization of the gene, upregulation of FDXR expression inhibited porcine epidemic diarrhea virus (PEDV) replication, significantly reducing both the in vitro PEDV replication level and viral titer. Therefore... FDXR The gene is a porcine epidemic diarrhea virus (PEDV) resistance gene, which can be used for certain antiviral applications (such as through effective activation). FDXR It can inhibit the replication of porcine epidemic diarrhea virus (PEDV) through gene expression; at the same time, it can provide highly efficient viral amplification cells for research on the pathogenic mechanism of PEDV (such as the molecular mechanism of regulating viral replication) and the preparation of PEDV vaccines (inactivated PEDV vaccines, attenuated vaccines, etc.), thus providing new ideas for the prevention and control of PEDV and having broad application prospects.

[0075] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0076] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

[0077] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. FDXR The application of gene expression activators in the preparation of products against porcine epidemic diarrhea virus infection, characterized in that, The FDXR The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The product is selected from any of the following: Cultures and raw materials used to combat the replication of porcine epidemic diarrhea virus; Drugs used to treat swine epidemic diarrhea virus infection or replication; A cell model resistant to swine epidemic diarrhea virus infection.

3. The application according to claim 2, characterized in that, The cell model against swine epidemic diarrhea virus infection was developed through the aforementioned... FDXR The cells were constructed by treating porcine cells with gene expression activators.

4. The application according to any one of claims 1 to 3, characterized in that, The FDXR Gene expression activators include FDXR Gene overexpression vectors, CRISPRa activation systems, or transcription activators.

5. The application according to claim 4, characterized in that, The FDXR Gene overexpression vectors include pcDNA3.1-FDXR.

6. The claim 1 FDXR The application of gene expression inhibitors in the preparation of products that promote porcine epidemic diarrhea virus infection, characterized in that, The product is selected from any of the following: Cultures used to promote the replication of porcine epidemic diarrhea virus and raw materials for vaccine production; Drugs that promote infection or replication of porcine epidemic diarrhea virus; A cell model that promotes porcine epidemic diarrhea virus infection.

7. The application according to claim 6, characterized in that, The cell model that promotes porcine epidemic diarrhea virus infection is obtained through the aforementioned... FDXR The cells were constructed by treating porcine cells with gene expression inhibitors.

8. The application according to claim 6 or 7, characterized in that, The FDXR The gene expression inhibitor is selected from any one or more of shRNA, siRNA, miRNA, antisense oligonucleotides, gene editing tools, and small chemical molecule inhibitors.

9. The application according to claim 8, characterized in that, The nucleotide sequence of the shRNA is shown in SEQ ID NO.

2.

10. The application according to claim 8, characterized in that, The gene editing tool is the CRISPR-Cas9 system, which includes a targeting mechanism. FDXR The sgRNA of a gene.

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