Application of IL-17A as a molecular target in inhibiting porcine transmissible gastroenteritis virus infection

Abstract

This invention belongs to the field of veterinary antiviral drug development and animal vaccine preparation technology, specifically involving the application of IL-17A as a molecular target in inhibiting porcine transmissible gastroenteritis virus (TGEV) infection. This invention found that after TGEV infection of cells, IL-17A expression was significantly upregulated and positively correlated with viral load: overexpression or addition of porcine IL-17A protein significantly promoted TGEV infection, while interference with IL-17A or the addition of corresponding inhibitors significantly inhibited TGEV infection. This invention screened antiviral drugs using IL-17A as a target and found that inhibiting IL-17A can significantly inhibit TGEV infection. Therefore, IL-17A plays an important role in resisting TGEV infection, providing a new target for the development of anti-TGEV drugs.

Description

Technical Field

[0001] This invention belongs to the field of veterinary antiviral drug development and animal vaccine preparation technology, specifically involving the application of IL-17A as a molecular target in inhibiting porcine transmissible gastroenteritis virus infection. Background Technology

[0002] Transmissible gastroenteritis virus (TGEV) is a single-stranded positive-sense RNA virus belonging to the genus Alphacoronavirus in the family Coronaviridae. It is the core pathogen causing acute, highly contagious gastrointestinal infectious diseases in pigs. The virus is primarily transmitted through the digestive tract. Infected pigs typically exhibit vomiting, severe watery diarrhea, and dehydration. The mortality rate in piglets under two weeks of age can reach 100%. Even surviving pigs often experience growth retardation and reduced feed efficiency, causing significant economic losses to the global pig industry.

[0003] TGEV recognizes and invades host cells using porcine aminopeptidase N (APN) as a receptor, with the intestine as its primary target organ. It can cause damage and necrosis of intestinal epithelial cells through oxidative stress, NF-κB-mediated apoptosis, and NLRP3 inflammasome activation-induced pyroptosis, leading to villus atrophy and disruption of the intestinal digestive and absorptive barrier. Current research confirms that TGEV infection can induce the massive release of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6, recruiting inflammatory cells such as neutrophils and macrophages to infiltrate the lamina propria of the intestinal mucosa, further aggravating histopathological damage. Simultaneously, TGEV can invade systemic immune organs such as the thymus, spleen, and lymph nodes, disrupting host immune homeostasis, causing immunosuppression, and easily leading to secondary mixed infections.

[0004] Currently, vaccination remains the core means of controlling TGEV, but existing vaccines suffer from problems such as delayed immune protection in newborn piglets and insufficient cross-protection against variant strains. Furthermore, the development of antiviral drugs targeting TGEV is still in its early stages, with a severe shortage of effective targets, and no anti-TGEV drugs targeting host immune regulatory factors have yet been approved for marketing. Therefore, screening and identifying key host regulatory molecules in the TGEV infection process and developing novel antiviral targets and intervention strategies are of significant theoretical and clinical value for the prevention and control of TGEV.

[0005] IL-17A (formerly known as CTLA-8) is a core member of the IL-17 cytokine family, primarily secreted by Th17 cells and CD8+. +T cells, γδ T cells, and natural killer T cells can also synthesize and secrete IL-17A. IL-17A, by binding to the IL-17RA / IL-17RC heterodimer receptor, activates multiple signaling pathways, including NF-κB, JAK / STAT, PI3K / Akt, and MAPK, inducing the expression of pro-inflammatory cytokines such as IL-6, IL-8, and TNF-α, as well as chemokines and matrix metalloproteinases. It is a key cytokine mediating the host's innate and adaptive immune responses and regulating inflammatory responses. Under physiological conditions, IL-17A-mediated inflammatory responses help the body resist pathogen invasion; however, under pathological conditions, excessive activation of IL-17A can disrupt the body's immune homeostasis, inducing excessive inflammatory responses and tissue pathological damage. Its role in autoimmune diseases, chronic inflammatory diseases, and infections by various pathogens has been widely confirmed.

[0006] Existing research shows that IL-17A plays an important regulatory role in various coronavirus infections: SARS-CoV-2 infection significantly upregulates IL-17A expression, and its level is positively correlated with the degree of lung inflammation and the risk of severe disease progression; in porcine epidemic diarrhea virus (PEDV) infection, excessive activation of IL-17A-mediated inflammatory pathways is an important factor exacerbating intestinal barrier damage. However, to date, the specific role and regulatory mechanism of IL-17A in TGEV infection remain unclear, and there are no reports, either domestically or internationally, on developing anti-TGEV infection drugs targeting IL-17A or on related technical solutions for enhancing the efficacy of TGEV vaccine production. Summary of the Invention

[0007] The purpose of this invention is to fill the technological gap in the existing technology where there is a lack of antiviral targets for TGEV and limited means to improve vaccine production efficiency. It clarifies for the first time the regulatory role of IL-17A in TGEV infection, provides the application of IL-17A as a novel target in the development of anti-TGEV infection drugs, and develops a technical solution for improving the production efficiency of TGEV vaccines based on IL-17A regulation.

[0008] This invention is the first to demonstrate that TGEV infection of host cells significantly upregulates the transcription and protein expression levels of IL-17A, and that the expression level of IL-17A is significantly positively correlated with the replication load of TGEV. Downregulating IL-17A expression using RNA interference technology or adding the IL-17A inhibitor Y-320 significantly inhibits TGEV replication in host cells; conversely, overexpression of porcine IL-17A or the addition of exogenous recombinant IL-17A significantly promotes TGEV replication. This invention screened antiviral drugs targeting IL-17A and found that inhibiting IL-17A significantly inhibits TGEV infection. Therefore, IL-17A plays an important role in combating TGEV infection, providing a new target for the development of anti-TGEV drugs.

[0009] To achieve the above objectives, the present invention provides the following solution: This invention provides the application of IL-17A protein or the gene encoding IL-17A protein as a drug target in the preparation of drugs for the prevention and / or treatment of TGEV infection, wherein the accession number of IL-17A protein is NP_001005729.1; and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.11.

[0010] This invention provides the application of IL-17A protein or the gene encoding IL-17A protein as a drug target in screening drugs for the prevention and / or treatment of TGEV infection, wherein the accession number of IL-17A protein is NP_001005729.1; and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.11.

[0011] This invention provides the use of reagents that inhibit or downregulate IL-17A expression or activity in the preparation of drugs for the prevention and / or treatment of TGEV infection.

[0012] Optionally, the reagent includes siRNA that specifically interferes with IL-17A gene expression and / or an IL-17A signaling pathway inhibitor; The sequence of the sense strand of the siRNA is shown in SEQ ID NO.5, and the sequence of the antisense strand is shown in SEQ ID NO.6; The IL-17A signaling pathway inhibitors include the IL-17A inhibitor Y-320.

[0013] More preferably, the drug targets IL-17A, and by inhibiting or downregulating the expression level or biological activity of IL-17A, it blocks the replication of TGEV in host cells, thereby achieving the prevention or treatment of TGEV infection.

[0014] The present invention provides a medicament for the prevention and / or treatment of TGEV infection, the medicament comprising an agent that inhibits or downregulates IL-17A expression or activity.

[0015] Optionally, the reagent includes siRNA that specifically interferes with IL-17A gene expression and / or an IL-17A signaling pathway inhibitor; The sequence of the sense strand of the siRNA is shown in SEQ ID NO.5, and the sequence of the antisense strand is shown in SEQ ID NO.6; The IL-17A signaling pathway inhibitors include the IL-17A inhibitor Y-320.

[0016] The siRNA described in this invention can efficiently silence the gene expression of IL-17A and significantly inhibit the replication of TGEV in host cells. It can be used as an active ingredient in the preparation of drugs against TGEV infection.

[0017] This invention provides the use of IL-17A expression promoters in any of the following: (1) Preparation of TGEV replication enhancer; (2) Preparation of host cell lines for TGEV vaccine production; (3) Prepare vaccines to prevent or treat TGEV infection.

[0018] Optionally, the IL-17A expression promoter includes an IL-17A gene overexpression vector and / or IL-17A protein; The nucleotide sequence of the IL-17A gene is shown in SEQ ID NO.11; the accession number of the IL-17A protein is NP_001005729.1.

[0019] More preferably, the TGEV replication enhancer increases the replication titer of TGEV in host cells by upregulating the expression and activity of IL-17A, and can be applied to the amplification of TGEV strains and the large-scale production of TGEV inactivated vaccines or live attenuated vaccines.

[0020] This invention provides a method for constructing an IL-17A protein overexpression cell line, including the step of transfecting host cells using an IL-17A gene overexpression vector; The nucleotide sequence of the IL-17A gene is shown in SEQ ID NO.11.

[0021] This invention provides the application of the IL-17A protein overexpressing cell line obtained by the above construction method in the preparation of host cell lines for TGEV vaccine production.

[0022] The present invention discloses the following technical effects: 1. This invention provides the first clear definition of the regulatory role of IL-17A in TGEV infection, offering a novel antiviral target: Through in vitro and in vivo experiments, this invention is the first to demonstrate that IL-17A is a key host regulatory factor in the TGEV infection process, and its expression level is positively correlated with TGEV replication efficiency. Inhibiting IL-17A can significantly block TGEV replication. This invention overcomes the limitations of existing anti-TGEV drug development that targets multiple viral proteins, providing the first anti-TGEV intervention target that targets the host immune regulatory factor IL-17A. This provides a new theoretical basis and technical direction for the development of novel anti-TGEV drugs, especially broad-spectrum anti-coronavirus drugs that target the host.

[0023] 2. Providing highly effective anti-TGEV intervention agents with broad clinical application prospects: The specific siRNA targeting IL-17A designed in this invention can efficiently silence IL-17A expression and significantly inhibit TGEV replication without affecting the normal physiological function of host cells; at the same time, it has been confirmed that the IL-17A inhibitor Y-320 can dose-dependently block TGEV proliferation. Both can be developed as active ingredients into anti-TGEV infection therapeutic drugs, or as feed additives for the prevention of TGEV infection in piglets, and have important clinical translational value.

[0024] 3. Develop new technologies to enhance the production efficiency of TGEV vaccines and improve vaccine production efficiency: This invention is the first to demonstrate that overexpression of IL-17A or the addition of exogenous IL-17A protein can significantly promote the replication of TGEV. Based on this, the IL-17A overexpression genetically engineered cell line developed can significantly increase the proliferation titer of TGEV, solving the industry pain points of low viral proliferation efficiency and insufficient antigen yield in the traditional vaccine production process. It can significantly reduce the production cost of TGEV vaccines, improve industrial production efficiency, and has important industrial application value. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 The results show the relative expression levels of IL-17A mRNA at different time points after TGEV infection of IPEC-J2 cells; Figure 2 The results of Western blot analysis of IL-17A and TGEV N protein after TGEV infection of IPEC-J2 cells; Figure 3Results of the study on the effect of targeted silencing of IL-17A on the transcriptional level of the TGEV N gene; Figure 4 The results of the detection of the effect of targeted silencing of IL-17A on the expression level of TGEV N protein; Figure 5 The results of the detection of the effect of the IL-17A inhibitor Y-320 on the transcriptional level of the TGEV N gene; Figure 6 The results show the effect of the IL-17A inhibitor Y-320 on the expression level of TGEV N protein; Figure 7 The results show the effect of IL-17A overexpression on the transcriptional level of the TGEV N gene; Figure 8 The results show the effect of IL-17A overexpression on TGEV N protein expression levels. Figure 9 The results show the effect of exogenous IL-17A recombinant protein on the transcriptional level of the TGEV N gene; Figure 10 The results show the effect of exogenous IL-17A recombinant protein on the expression level of TGEV N protein. Detailed Implementation

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0030] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0031] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0032] 1. Strains and cells: The porcine small intestinal epithelial cell line IPEC-J2 was preserved by the Animal Molecular Pathogen Laboratory of Henan Agricultural University; the TGEV HN-2012 strain was isolated, identified and preserved by the Animal Molecular Pathogen Laboratory of Henan Agricultural University (it has been published in the literature "Genetic variation analysis of M gene and its eukaryotic expression study of TGEV HN-2012 isolate").

[0033] 2. Main reagents: DMEM basal medium, fetal bovine serum (FBS), and Opti-MEM serum-free medium were purchased from Gibco; Trizol RNA extraction reagent, reverse transcription kit, and ChamQ Universal SYBR qPCR Master Mix were purchased from Nanjing Novizan Biotechnology Co., Ltd.; Lipomaster 3000 transfection reagent was purchased from Shanghai Yisheng Biotechnology Co., Ltd.; PCAGGS-HA-IL-17A eukaryotic overexpression vector was synthesized by Sangon Biotech (Shanghai) Co., Ltd.; IL-17A specific siRNA and negative control siRNA were synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd.; IL-17A inhibitor Y-320 was purchased from Selleck Chemicals; porcine IL-17A recombinant protein (NP_001005729.1) was kindly provided by the Veterinary Research Institute of Jiangsu Academy of Agricultural Sciences and is disclosed in Table 1 of the literature "Preparation and functional identification of various porcine cytokines", where its name is IL-17α; anti-IL-17A antibody, anti-TGEV antibody... N protein antibody, anti-β-actin antibody, and HRP-labeled goat anti-rabbit / mouse secondary antibody were all purchased from Cell Signaling Technology; RIPA protein lysis buffer and ECL chemiluminescence reagent were purchased from Beijing Beyotime Biotechnology Co., Ltd.

[0034] 3. Major instruments: CO2 constant temperature cell incubator (Thermo Fisher Scientific); real-time quantitative PCR instrument (Bio-Rad CFX96); protein electrophoresis and wet transfer system (Bio-Rad); ECL chemiluminescence imaging system (Shanghai Tianneng Technology Co., Ltd.); high-speed refrigerated centrifuge (Eppendorf).

[0035] Example 1: Effect of TGEV infection on IL-17A expression in host cells This embodiment uses real-time quantitative PCR (RT-qPCR) and Western blot techniques to detect changes in IL-17A transcription and protein expression levels in IPEC-J2 cells after TGEV infection, clarifying the correlation between IL-17A and TGEV infection. The specific steps are as follows: 1. Cell Culture and Viral Infection After resuscitation, IPEC-J2 cells were passaged in DMEM complete medium (DMEM basal medium + 10% FBS + 1% penicillin-streptomycin) containing 10% FBS and 1% penicillin-streptomycin antibiotics at 37°C and 5% CO2 saturated humidity. IPEC-J2 cells in the logarithmic growth phase were then cultured at a rate of 1×10⁻⁶ cells / cell. 5 Cells were seeded at a density of 1 cell / well in 24-well cell culture plates and cultured until 90% confluence. The culture medium was then discarded, and TGEV HN-2012 virus solution was seeded at a multiplicity of infection (MOI) of 1. The plate was gently agitated to ensure even coverage of the cells with the virus solution. After 1 h of adsorption, the virus solution was discarded, and the cells were gently washed three times with sterile D-Hanks buffer. 500 μL of DMEM maintenance medium containing 2% FBS was added to each well (DMEM basal medium + 2% FBS), and the cells were cultured further. Cell samples were collected at 12 h and 24 h post-infection. A blank control group (uninfected with the virus) was also included, with three biological replicates for each group.

[0036] 2. RT-qPCR detection of IL-17A mRNA expression level Total RNA was extracted from cells in each group using the Trizol method. RNA concentration and purity were measured using an ultra-micro spectrophotometer to ensure OD... 260 / OD 280 The ratio was between 1.8 and 2.0. Following the reverse transcription kit instructions, 1 μg of total RNA was reverse transcribed into cDNA. Using cDNA as a template, the relative expression level of IL-17A mRNA was detected by RT-qPCR, with porcine β-actin gene used as an internal reference gene.

[0037] The primer sequences are as follows: β-actin-F: 5'-TTCCAGCCCTCCTTCCTG-3', SEQ ID NO.1; β-actin-R: 5'-AGGTCCTTGCGGATGTCG-3', SEQ ID NO.2; IL-17A-F: 5'-CAATTCCCGTGGTCGGAAGA-3', SEQ ID NO.3; IL-17A-R: 5'-TTTACGTTGGCCCTTCACCA-3', SEQ ID NO. 4.

[0038] RT-qPCR reaction system (20 μL): 10 μL 2×SYBR qPCR Master Mix, 0.4 μL each of upstream and downstream primers (10 μmol / L), 2 μL cDNA template, and 7.2 μL enzyme-free water.

[0039] Reaction program: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 10 s, 60℃ annealing for 30 s, for a total of 40 cycles; Melting curve analysis: fluorescence signal was collected every 0.5℃ from 65℃ to 95℃.

[0040] Use 2 -ΔΔCt The relative expression levels of IL-17A mRNA were calculated using the t-test, and statistical analysis was performed. P < 0.05 was considered statistically significant, and P < 0.01 was considered extremely statistically significant.

[0041] Test results as follows Figure 1 As shown, compared with the blank control group, the relative expression level of IL-17A mRNA was significantly increased 12 h after TGEV HN-2012 infection of IPEC-J2 cells (P<0.01), and further upregulated after 24 h of infection. Moreover, its expression level was positively correlated with the TGEV HN-2012 infection time, indicating that TGEV HN-2012 infection can significantly promote the transcription of IL-17A gene in host cells.

[0042] 3. Western blot detection of IL-17A protein expression level Cells from each group were collected, and RIPA protein lysis buffer containing 1 mmol / L PMSF was added. After lysis on ice for 30 min, the cells were centrifuged at 12000 r / min for 15 min at 4 °C. The supernatant was collected, and the protein concentration was determined using the BCA method. An equal volume of protein sample was taken, and 5×SDS protein loading buffer was added. The sample was heated in a boiling water bath for 10 min to completely denature the protein. After separation by SDS-PAGE gel electrophoresis, the proteins were wet-transferred onto a PVDF membrane and blocked with 5% skim milk at room temperature for 2 h. Primary antibodies (anti-IL-17A antibody 1:1000, anti-TGEV N protein antibody 1:1000, anti-β-actin antibody 1:2000) were added and incubated overnight at 4°C. The membrane was washed three times with TBST for 10 min each time, and HRP-labeled secondary antibody (1:5000 dilution) was added and incubated at room temperature for 1 h. After washing the membrane three more times with TBST, ECL chemiluminescence reagent was added, and the membrane was exposed and imaged in a chemiluminescence imaging system. The gray values ​​of the protein bands were analyzed using β-actin as an internal control.

[0043] Test results as follows Figure 2 As shown, compared with the blank control group, the expression level of IL-17A protein in IPEC-J2 cells was significantly upregulated after TGEV HN-2012 infection, and was positively correlated with the expression level of TGEV N protein, further confirming that TGEV HN-2012 infection can significantly activate the expression of IL-17A in host cells, and its expression level is closely related to viral replication efficiency.

[0044] Example 2: Inhibitory effect of targeted silencing of IL-17A on TGEV replication This embodiment uses RNA interference technology to target and silence the expression of IL-17A in IPEC-J2 cells, and examines its effect on TGEV replication to verify the feasibility of IL-17A as an anti-TGEV target.

[0045] 1. siRNA sequence design and synthesis A specific siRNA was designed targeting the coding region of the porcine IL-17A gene, and a negative control siRNA (siNC) without homologous sequences was also designed. The sequences are as follows: IL-17A-siRNA positive strand: 5'-GGAAGGAAGAUCAUCACAUTT-3', SEQ ID NO.5; IL-17A-siRNA antisense strand: 5'-AUGUGAUGAUCUUCCUUCCTT-3', SEQ ID NO.6; siNC Justice Chain: UUCUCCGAACGUGUCACGUTT, SEQ ID NO.7; siNC antisense chain: ACGUGACACGUUCGGAGAATT, SEQ ID NO.8.

[0046] 2. Cell transfection and viral infection IPEC-J2 cells in the logarithmic growth phase were harvested at a concentration of 1×10⁻⁶. 5 Seeds were placed into 24-well cell culture plates at a density of cells / well. When cell confluence reached 60%–70%, siRNA transfection was performed. The transfection system was as follows: In tube A, 25 μL of Opti-MEM serum-free medium and 3 μL of Lipomaster 3000 transfection reagent were added and gently mixed; in tube B, 25 μL of Opti-MEM serum-free medium and 30 pmol IL-17A-siRNA / siNC were added and gently mixed; the solution from tube B was added dropwise to tube A, gently mixed, and incubated at room temperature for 15 min to form the transfection complex. The transfection complex was then added dropwise to the cell culture wells, the plate was gently shaken to mix, and the plates were incubated for further culture.

[0047] Twelve h after transfection, the medium containing the transfection complex was discarded, and the cells were washed twice with pre-warmed D-Hanks buffer. The medium was then replaced with DMEM maintenance medium containing 2% FBS and cultured for another 24 h. Subsequently, TGEV HN-2012 virus solution was inoculated at MOI = 1, and after 1 h of adsorption, the DMEM maintenance medium was replaced. Cell samples were collected after another 24 h of culture. The experiment included a blank control group (untransfected, uninfected, control), a TGEV-infected group (TGEV), a TGEV-infected control group (transfected with siNC+TGEV, HN-2012 infection, TGEV+siNC), and an IL-17A silencing group (transfected with IL-17A-siRNA+TGEV, HN-2012 infection, TGEV+siIL-17), with three biological replicates for each group.

[0048] 3. RT-qPCR detection of TGEV N gene transcription level Total RNA was extracted from cells and reverse transcribed into cDNA according to the method in Example 1. The relative expression level of TGEV N gene mRNA was detected by RT-qPCR, with β-actin as an internal reference gene.

[0049] The primer sequences for the TGEV N gene are as follows: TGEV NF: 5'-CTCGTGAAGGCGGGAATCAT-3', SEQ ID NO.9; TGEV NR: 5'-TGGAGAGTCCATGGTGAGGT-3', SEQ ID NO. 10.

[0050] The RT-qPCR reaction system and procedure are the same as in Example 1, using 2 -ΔΔCt The relative expression level of the TGEV N gene was calculated using this method.

[0051] Test results as follows Figure 3 As shown, compared with the TGEV-infected control group, the relative mRNA expression level of the TGEV N gene in IPEC-J2 cells was significantly reduced after targeted silencing of IL-17A (P<0.01), indicating that downregulating IL-17A expression can significantly inhibit TGEV gene transcription.

[0052] 4. Western blot analysis of TGEV N protein expression levels Total cellular protein was extracted according to the method in Example 1, and the expression level of TGEV N protein was detected by Western blot, with Tubulin as an internal control.

[0053] Test results as follows Figure 4 As shown, compared with the TGEV-infected control group, the expression level of TGEV N protein in the IL-17A-silenced group was significantly downregulated, further confirming that targeted silencing of IL-17A can significantly inhibit TGEV replication in host cells.

[0054] Example 3: Inhibitory effect of IL-17A inhibitor Y-320 on TGEV replication In this embodiment, IPEC-J2 cells were treated with the IL-17A-specific inhibitor Y-320, and the effect on TGEV replication was detected to verify the anti-TGEV effect of targeting and inhibiting IL-17A activity.

[0055] IPEC-J2 cells in the logarithmic growth phase were harvested at a concentration of 1×10⁻⁶. 5 Cells were seeded at a density of 1 cell / well in 24-well cell culture plates and cultured until 90% confluence. Y-320 was then added at final concentrations of 2.5 nmol / L, 25 nmol / L, and 250 nmol / L, respectively, and labeled as TGEV + 2.5 nmol / L, TGEV + 25 nmol / L, and TGEV + 250 nmol / L. A TGEV-only infection control group (TGEV) and a blank control group (uninfected and untreated) were also included, with three biological replicates for each group. After 24 h of drug treatment, TGEV HN-2012 virus solution was inoculated at an MOI of 1. After 1 h of adsorption, the medium was replaced with maintenance medium containing the corresponding concentration of Y-320. Cells were cultured for another 24 h, and cell samples were collected. The transcription and protein expression levels of the TGEV N gene were detected by RT-qPCR and Western blot, respectively, using the same experimental methods as in Examples 1 and 2, with Tubulin as an internal control.

[0056] RT-qPCR test results are as follows Figure 5 As shown, compared with the TGEV-infected control group, Y-320 treatment significantly reduced the mRNA expression level of the TGEV N gene in a dose-dependent manner, with the most significant inhibitory effect at a concentration of 250 nmol / L. P <0.01).

[0057] Western blot results are as follows Figure 6 As shown, Y-320 treatment significantly downregulated the expression level of TGEV N protein, and the inhibitory effect was significantly dose-dependent, confirming that inhibiting the biological activity of IL-17A can effectively block the replication of TGEV in host cells.

[0058] Example 4: The promoting effect of IL-17A overexpression on TGEV replication In this embodiment, porcine IL-17A was overexpressed in IPEC-J2 cells using a eukaryotic overexpression vector, and its effect on TGEV replication was examined to verify the synergistic potential of IL-17A in TGEV vaccine production.

[0059] IPEC-J2 cells in the logarithmic growth phase were harvested at a concentration of 1×10⁻⁶. 5 Seeds were placed into 24-well cell culture plates at a density of 10 cells / well. When cell confluence reached 60%–70%, plasmid transfection was performed. The transfection system was as follows: In tube A, 25 μL of Opti-MEM serum-free medium and 3 μL of Lipomaster 3000 transfection reagent were added and gently mixed. In tube B, 25 μL of Opti-MEM serum-free medium and 1 μg of pCAGGS-HA-IL-17A overexpression plasmid (based on pCAGGS-HA, with the plasmid added at the restriction enzyme sites) were added. EcoR I and XhoThe IL-17A coding gene was inserted between the 1 and 2, resulting in the pCAGGS-HA-IL-17A overexpression plasmid. The nucleotide sequence of the IL-17A coding gene is shown in SEQ ID NO.11, specifically: gaattcatgTACCCATACGATGTTCCAGATTACGCTactcctgtgagatcctcgtccctgtcactgctgcttctgctgagcctggtggctctcgtgaaggcgggaatcatgatcccacaaagtccaggatgcccaaaaactgaggacaagaacttccctcagcatgtaagggtcaacttgaacatcttgaaccggagcacacctgccagacggccctcagattactccaaacgcttcacctcacca The solution in tube B was mixed with 2 μL of P3000 enhancement reagent and gently stirred. The solution in tube B was then added dropwise to tube A, mixed thoroughly, and allowed to stand at room temperature for 15 min to form a transfection complex. The transfection complex was then added to cell culture wells, and three biological replicates were set up for each group: a group transfected with the empty vector pCAGGS-HA, a control group (untransfected), and a TGEV-infected group.

[0060] After 12 h of transfection, the maintenance medium was replaced and cultured for another 24 h. Then, TGEV HN-2012 virus solution was inoculated at MOI = 1. After 1 h of adsorption, the maintenance medium was replaced and cultured for another 24 h. Cell samples were collected and the transcription and protein expression levels of the TGEV N gene were detected by RT-qPCR and Western blot, respectively. The experimental methods were the same as in Examples 1 and 2, with Tubulin as an internal control.

[0061] RT-qPCR test results are as follows Figure 7As shown, compared with the control group transfected with empty vector, the relative mRNA expression level of the TGEV N gene in IPEC-J2 cells was significantly increased after overexpression of IL-17A. P <0.01).

[0062] Western blot results are as follows Figure 8 As shown, the expression level of TGEV N protein in cells overexpressing IL-17A was significantly upregulated compared with that in the control group, confirming that overexpression of IL-17A can significantly promote the replication of TGEV in host cells.

[0063] Example 5: The promoting effect of exogenous IL-17A recombinant protein on TGEV replication This embodiment verifies the feasibility of exogenous IL-17A as a TGEV replication enhancer by adding exogenous IL-17A recombinant protein to cell culture medium and examining its effect on TGEV replication.

[0064] IPEC-J2 cells in the logarithmic growth phase were harvested at a concentration of 1×10⁻⁶. 5 Cells were seeded at a density of 1 cell / well in 24-well cell culture plates and cultured until 90% confluence. Porcine IL-17A recombinant protein was then added at final concentrations of 1 ng / mL, 10 ng / mL, and 100 ng / mL, respectively. A TGEV infection control group and a blank control group without protein were also included, with three biological replicates for each group. After 24 h of protein treatment, TGEV HN-2012 virus solution was inoculated at an MOI of 1. After 1 h of adsorption, the medium was replaced with maintenance medium containing the corresponding concentration of IL-17A recombinant protein. Cells were cultured for another 24 h, and then cell samples were collected. The transcription and protein expression levels of the TGEV N gene were detected by RT-qPCR and Western blot, respectively, using the same experimental methods as in Examples 1 and 2, with Tubulin as an internal control.

[0065] RT-qPCR test results are as follows Figure 9 As shown, compared with the TGEV-infected control group, exogenous IL-17A recombinant protein significantly increased the mRNA expression level of the TGEV N gene in a dose-dependent manner, with the most significant promoting effect at a concentration of 100 ng / mL. P <0.01).

[0066] Western blot results are as follows Figure 10 As shown, treatment with exogenous IL-17A recombinant protein can significantly upregulate the expression level of TGEVN protein, and the promoting effect is significantly dose-dependent, confirming that exogenous IL-17A recombinant protein can be used as a TGEV replication enhancer and applied in the process of TGEV strain amplification and vaccine production.

[0067] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. IL-17A protein or the coding gene of IL-17A protein as a drug target in the preparation of a drug for preventing and / or treating TGEV infection, characterized in that, The accession number for the IL-17A protein is NP_001005729.1; the nucleotide sequence of the encoding gene is shown in SEQ ID NO.

11.

2. The use of IL-17A protein or the encoding gene of IL-17A protein as a drug target in screening drugs for preventing and / or treating TGEV infection, characterized in that, The accession number for the IL-17A protein is NP_001005729.1; the nucleotide sequence of the encoding gene is shown in SEQ ID NO.

11.

3. The use of reagents that inhibit or downregulate IL-17A expression or activity in the preparation of drugs for the prevention and / or treatment of TGEV infection.

4. Use according to claim 3, characterized in that, The reagents include siRNA that specifically interferes with IL-17A gene expression and / or IL-17A signaling pathway inhibitors; The sequence of the sense strand of the siRNA is shown in SEQ ID NO.5, and the sequence of the antisense strand is shown in SEQ ID NO.6; The IL-17A signaling pathway inhibitors include the IL-17A inhibitor Y-320.

5. A medicament for preventing and / or treating TGEV infection, characterized by, The drug includes agents that inhibit or downregulate IL-17A expression or activity.

6. The medicament according to claim 5, characterized in that, The reagents include siRNA that specifically interferes with IL-17A gene expression and / or IL-17A signaling pathway inhibitors; The sequence of the sense strand of the siRNA is shown in SEQ ID NO.5, and the sequence of the antisense strand is shown in SEQ ID NO.6; The IL-17A signaling pathway inhibitors include the IL-17A inhibitor Y-320.

7. The application of IL-17A expression promoters in any of the following: (1) Preparation of TGEV replication enhancer; (2) Preparation of host cell lines for TGEV vaccine production; (3) Prepare vaccines for the prevention and / or treatment of TGEV infection.

8. Use according to claim 7, characterized in that, The IL-17A expression promoter includes an IL-17A gene overexpression vector and / or IL-17A protein; The nucleotide sequence of the IL-17A gene is shown in SEQ ID NO.11; the accession number of the IL-17A protein is NP_001005729.

1.

9. A method for constructing a cell line overexpressing IL-17A protein, characterized in that, This includes the step of transfecting host cells using an IL-17A gene overexpression vector; The nucleotide sequence of the IL-17A gene is shown in SEQ ID NO.

11.

10. The application of the IL-17A protein overexpressing cell line obtained by the construction method of claim 9 in the preparation of host cell lines for TGEV vaccine production.

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