SiRNA targeting nsp9 of porcine epidemic diarrhea virus and antiviral application thereof

Abstract

The application discloses a kind of siRNA and antiviral application of targeting swine epidemic diarrhea virus Nsp9, belong to the field of biotechnology.The application designs a kind of siRNA targeting PEDV Nsp9, the nucleotide sequence of the siRNA sense strand is as shown in SEQ ID NO.3, the nucleotide sequence of the siRNA antisense strand is as shown in SEQ ID NO.4.It is proved by cell transfection experiment and virus infection experiment that the siRNA prepared in the application can significantly reduce the replication infection of PEDV in cells, effectively inhibit the expression of virus, and play an antiviral role;And the siRNA has no cytotoxicity, and has extensive application prospect.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a siRNA targeting porcine epidemic diarrhea virus Nsp9 and its antiviral application. Background Technology

[0002] Porcine epidemic diarrhea virus (PEDV) is a coronavirus that causes diarrhea, vomiting, dehydration, and high mortality in suckling piglets, seriously threatening the healthy development of the pig industry. PEDV can infect pigs of different ages, but suckling piglets, especially newborn piglets, experience the most severe symptoms after infection, with a mortality rate as high as 100%.

[0003] PEDV is an enveloped, pleomorphic viral particle with a diameter of approximately 95–190 nm (including spikes). The PEDV genome is approximately 28 kb in length, encoding four structural proteins: spike protein (S), small membrane protein (E), membrane glycoprotein (M), and nucleocapsid protein (N), 16 non-structural proteins (nsp1–nsp16), and one accessory protein, ORF3. Nsp9 The Nsp9 protein, with a gene length of approximately 324 bp, possesses nucleotide-binding properties and plays a crucial role in the formation of the viral replication complex. Chinese researchers have resolved the crystal structure of the Nsp9 protein and constructed a series of mutants, demonstrating that the positively charged amino acids on the Nsp9 protein surface play a vital role in nucleotide binding.

[0004] Small interfering RNA (siRNA) is a double-stranded RNA molecule composed of 20-25 nucleotides that precisely silences the expression of specific genes through RNA interference mechanisms. siRNA was first discovered in 1999 by David Baulcomb's laboratory. In 2001, Thomas Tuschl's team synthesized 21 nt siRNA, achieving the first successful silencing of a target gene in human cells (HeLa cells), laying the foundation for siRNA therapy. Based on siRNA's precise, efficient, and broad-spectrum gene silencing capabilities, this technology has been widely developed for the treatment of various diseases, including viral infections, genetic diseases, metabolic diseases, and cancer. Since the launch of the first siRNA drug (Patisiran) in 2018, and with recent breakthroughs in hypertension and oncology, advancements in delivery technology and chemical modification are driving the expansion of siRNA into multiple disease areas, potentially making it the third major pillar of biotechnology after monoclonal antibodies and vaccines.

[0005] PEDV infection damages the epithelial cells of the small intestine in piglets, causing malabsorption of nutrients in the digestive tract, leading to diarrhea, vomiting, and dehydration. The mortality rate of newborn piglets infected with PEDV can reach 100%, seriously jeopardizing the healthy development of the pig farming industry. However, due to the... S The mutagenic nature of genes makes it difficult for traditional vaccines to provide effective immune protection. Based on this problem, this invention aims to develop a safe and effective novel siRNA drug against PEDV, utilizing the key mechanistic role of PEDV Nsp9 in viral replication. Summary of the Invention

[0006] The purpose of this invention is to provide a siRNA targeting porcine epidemic diarrhea virus (PEDV) Nsp9 and its antiviral application, in order to solve the problems existing in the prior art. This invention designs a siRNA targeting PEDV Nsp9, which acts on PEDV-infected Vero E6 kidney epithelial cells of African green monkeys. Experimental verification shows that the siRNA targeting PEDV Nsp9 can effectively inhibit PEDV infection and exert a highly efficient antiviral effect; moreover, this siRNA is non-cytotoxic and has broad application prospects.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] This invention provides an siRNA that resists porcine epidemic diarrhea virus.

[0009] The nucleotide sequence of the positive strand of the siRNA is: GCAUAGUUGGAGAAGGUAATT;

[0010] The antisense strand nucleotide sequence of the siRNA is: UUACCUUCUCCAACUAUGCTT;

[0011] The siRNA targets non-structural protein 9 of porcine epidemic diarrhea virus.

[0012] The present invention also provides the application of the siRNA in the preparation of porcine epidemic diarrhea virus inhibitors.

[0013] The present invention also provides a porcine epidemic diarrhea virus inhibitor, the inhibitor comprising the siRNA of claim 1.

[0014] The present invention also provides the use of the siRNA in the preparation of a medicament for treating diseases caused by porcine epidemic diarrhea virus infection.

[0015] The present invention also provides a drug for treating diseases caused by porcine epidemic diarrhea virus infection, the drug comprising the siRNA described above.

[0016] Preferably, the dosage form of the drug is capsule, tablet, granule, emulsion, pill, or oral liquid.

[0017] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0018] Preferably, the excipients include binders, disintegrants, fillers, stabilizers, preservatives, coating materials, and / or fragrances.

[0019] The present invention also provides the use of a host cell containing the siRNA in the preparation of a vaccine to prevent porcine epidemic diarrhea virus disease.

[0020] The present invention discloses the following technical effects:

[0021] This invention provides an siRNA targeting porcine epidemic diarrhea virus (PEDV) non-structural protein 9 (Nsp9) and its antiviral application. This siRNA is based on PEDV... Nsp9 Genes were designed and optimized based on secondary structure prediction results, ultimately yielding a set of siRNAs that inhibit PEDV replication. Cell transfection and viral infection experiments confirmed that the siRNAs prepared in this invention can significantly reduce PEDV replication and infection in cells, effectively inhibiting viral expression, providing new ideas and insights for the development of novel anti-PEDV drugs. Attached Figure Description

[0022] 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.

[0023] Figure 1 Genetic evolution analysis of the whole genome sequence of the strains isolated and preserved in our laboratory;

[0024] Figure 2 Analysis of the conservation of Nsp9 amino acid sequence in different PEDV subtypes;

[0025] Figure 3 A diagram showing the predicted secondary structure of PEDV NSP9 RNA using RNAfold WebServer;

[0026] Figure 4 A: The inhibitory effect of Nsp9-targeting siRNA on PEDV infection; A: The effect of siNC and siNsp9-59 transfection on PEDV infection. Nsp9A: Inhibitory effect of gene; B: Inhibitory effect of siNC and siNsp9 on PEDV infection detected by Western blot; C: Inhibitory effect of siNC and siNsp9 on PEDV infection detected by qPCR.

[0027] Figure 5 The effect of siRNA targeting Nsp9 on cell viability. Detailed Implementation

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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 readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0032] 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.

[0033] Example 1

[0034] 1. Experimental Materials

[0035] 1.1 Cells and viruses used in this invention: The cells used in in vitro experiments were the VeroE6 cell line of African green monkey kidney epithelial cells, preserved in our laboratory; the porcine epidemic diarrhea virus strains CV777 (GIb subtype), SDZB (GIIb subtype), SDLY (GIIb subtype), SDJNing (GIIc subtype), and SDQD (GIIc subtype) (Zhang Zhihao, Li Jianda, Cao Zhi, et al. Molecular epidemiological survey of porcine epidemic diarrhea virus in Shandong Province and isolation and identification of epidemic strains [J]. Animal Husbandry and Veterinary Medicine, 2025, 57(7):80-88.) were isolated and preserved by the Key Laboratory of Livestock and Poultry Biological Omics of the Ministry of Agriculture and Rural Affairs, and are open to researchers in this field. The whole genome sequence genetic evolution analysis of the strains isolated and preserved in our laboratory, such as Figure 1 As shown.

[0036] 1.2 Main reagents and materials used in this invention: DMEM medium, Opti-MEM serum-depleted medium, 0.25% trypsin (Gibco); fetal bovine serum (BI); Cell Counting Kit-8 (CCK8) kit (MCE); trypsin (Sigma); Lipofectamine™ RNAiMAX transfection reagent (Thermo Fisher); mouse-derived PEDV-N protein monoclonal antibody (Shandong Academy of Agricultural Sciences, Institute of Animal Husbandry and Veterinary Medicine); HRP-labeled GAPDH antibody, HRP-labeled goat anti-mouse secondary antibody; RNA extraction reagent RNA-easy Isolation Reagent, RNA reverse transcription reagent HiScript III RT SuperMix for qPCR, real-time PCR reagent ChamQ Universal SYBR qPCR Master Mix (Novizan); ultrasensitive ECL chemiluminescence solution BeyoECL Moon (Beyotime).

[0037] 2. Experimental Methods

[0038] 2.1 Differential analysis and secondary structure prediction of the amino acid sequence (SEQ ID NO.1) of porcine epidemic diarrhea virus (PEDV) non-structural protein 9 (Nsp9): The differences in the Nsp9 protein sequence among different PEDV subtype strains (tested strains are shown in Table 1) were compared using MEGA6 software; the secondary structure of PEDV Nsp9 RNA was predicted using the online software RNAfold WebServer.

[0039] The amino acid sequence (SEQ ID NO.1) of porcine epidemic diarrhea virus (PEDV) nonstructural protein 9 (Nsp9) is as follows:

[0040] NNEIIPGKLKQRSIKAEGDGIVGEGKALYNNEGGRTFMYAFISDKPDLRVVKWEFDGGCNTIELEPPRKFLVDSPNGAQIKYLYFVRNLNTLRRGAVLGYIGATVRLQ.

[0041] The nucleotide sequence (SEQ ID NO.2) of porcine epidemic diarrhea virus (PEDV) nonstructural protein 9 (Nsp9) is as follows:

[0042] aataatgagattattcctggtaagctgaagcagcgctccattaaggcagaaggagatggcatagttggagaaggtaaggcactttacaataatgagggtggacgtacttttatgtatgctttcatttcagataaaccggacctgcgtgtagttaagtggggag ttcgatggtggttgtaacactattgagctagaaccaccacgtaagttcttggtggattctcctaatggtgcacagatcaagtatctctactttgttcgtaaccttaacacgttgcgtaggggtgctgttcttggctacataggtgccactgtacgcttgcag.

[0043] 2.2 siRNA design based on the porcine epidemic diarrhea virus (PEDV) nonstructural protein 9 (Nsp9) gene (SEQ ID NO.2): Based on the CDS region of the PEDV CV777 strain (accession number: KT323979) Nsp9 gene, sequences targeting PEDV were designed using the online software BLOCK-iT RNAi Designer (Invitrogen, https: / / rnaidesigner.thermofisher.com / rnaiexpress / ) with a GC content close to 50%, no more than 4 consecutive repeats of a single base, and a length of 19-21 bases. Nsp9 The siNsp9-59 of the gene (Table 1). The negative control siRNA (siNC) was designed with the exact same base composition as siNsp9-59. After the bases were randomly arranged, Blast alignment was performed. After confirming that the sequence had no homology with known genes in the PEDV and Genebank databases, it could be used as siNC (Table 1).

[0044] Table 1. siRNA sequence information targeting PEDV Nsp9

[0045]

[0046] 2.3 Cell Transfection: Vero cells were seeded in 12-well cell culture plates, and siNsp9-59 and siNC transfection groups were set up. The plates were incubated at 37℃ with 5% CO2. When the cells reached 70% confluence, the supernatant was discarded, and 800 μL of serum-free DMEM medium was added for incubation for 1-2 h for siRNA transfection. Transfection solutions A and B were prepared according to the Lipofectamine™ RNAiMAX transfection reagent instructions: Solution A: 6 μL of Lipofectamine™ RNAiMAX transfection reagent was added to 100 μL of Opti-MEM medium; Solution B: 20 ​​pmol of siNsp9-59 and 20 pmol of siNC were added to 100 μL of Opti-MEM medium, respectively. Solutions A and B were gently mixed and then added to the cells, and the cells were cultured for 18-24 h for PEDV infection and cell viability detection.

[0047] 2.4 PEDV Infection: Transfected cells were inoculated with CV777 strain (GIb subtype), SDZB strain (GIIb subtype), SDLY strain (GIIb subtype), SDJNing strain (GIIc subtype), and SDQD strain (GIId subtype) at a MOI of 0.1 and incubated at 37°C in a 5% CO2 incubator for 1 h. After incubation, the virus solution was discarded, and residual virus solution was removed by rinsing with PBS. The cells were then cultured in maintenance medium (DEME medium containing 2% FBS) for 24 h. For SDZB, SDLY, SDJNing, and SDQD strains, trypsin was added to a final concentration of 10 μg / mL during virus incubation and in the maintenance medium.

[0048] 2.5 Quantitative Real-Time PCR (qPCR) Detection of Nsp9 Expression and Viral Load: Total RNA was extracted from cells using the Trizol method, and reverse transcription was performed using Novizan's HiScript II Reverse Transcriptase kit to obtain cDNA from the cell samples. qPCR detection was performed according to Novizan's ChamQ SYBR qPCR Master Mix reagent instructions. Nsp9 Genes and N The primers for detecting the target gene and the internal reference gene are shown in Table 2. Each of the target gene and the internal reference gene was tested three times in duplicate, using 2... −ΔΔCT Analyze the experimental data.

[0049] Table 2 Primer Sequences

[0050]

[0051] 2.4 Western blot detection of PEDV N protein expression: Cell samples were lysed in RIPA lysis buffer, centrifuged, and the supernatant was collected. Protein concentration was determined according to the BCA protein concentration assay kit instructions. Sample proteins were diluted and adjusted according to concentration, and after SDS denaturation, SDS-PAGE gel electrophoresis was performed. Subsequently, the protein was transferred to a methanol-activated PVDF membrane using a rapid wet transfer apparatus. After blocking with 5% skim milk powder for 2 h, the PVDF membrane containing the target band was incubated overnight at 4°C with mouse PEDV-N protein monoclonal antibody (1:1000) and HRP-labeled GAPDH antibody (1:5000), respectively. After 5 washes with TBST, HRP-labeled goat anti-mouse secondary antibody (1:8000) was added. After washing, the membrane was exposed and observed using an automated chemiluminescence image analysis system with ECL chemiluminescence buffer. The obtained protein bands were analyzed using ImageJ software.

[0052] 2.5 Plaque formation assay to detect viral titer in supernatant: Supernatant from siRNA-transfected and PEDV-infected samples was collected and serially diluted 10-fold to 10⁻⁶. -1 -10 -5 Gradient inoculum was seeded into Vero cells that had grown into a dense monolayer and incubated at 37°C with 5% CO2 for 1 h. After incubation, the viral load was discarded, and the cells were washed with PBS to remove residual viral load. A 1:1 mixture of LMP low-melting-point agarose gel and 2×DMEM was added to the cell culture plates, and after solidification, the plates were incubated for 48-60 h. Once plaques appeared, the cells were fixed with 4% paraformaldehyde for 20 minutes, then the gel in the wells was discarded, and the cells were incubated with 0.1% crystal violet at room temperature for 2 h. After washing, the cells were photographed and counted.

[0053] 2.6 Effect of CCK8 assay on cell viability: The effect of siNsp9-59 transfection on cell viability was detected according to the MedChemExpress CCK8 reagent instructions; cells transfected with siNC served as the negative control group.

[0054] 3. Experimental Results

[0055] 3.1 Conservatism Analysis and Secondary Structure Prediction of PEDV Nsp9

[0056] This invention compared the Nsp9 amino acid sequences of representative strains of different PEDV subtypes using MEGA6 software and found that Nsp9 is highly conserved in different PEDV subtypes. Figure 2This provides targets for discovering broad-spectrum anti-PEDV drugs; the secondary structure of PEDV Nsp9 RNA was predicted using the online software RNAfold WebServer. Figure 3 This provides an open region that effectively combines the hit rate and silencing efficiency of siRNA screening.

[0057] 3.2 Effects of siNsp9-59 transfection on PEDV infection

[0058] To investigate whether the siNsp9-59 designed in this invention can inhibit PEDV replication and infection, siNsp9-59 and siNC were transfected into Vero cells at a concentration of 30 pmol. After incubation for 18 h, the cells were inoculated with CV777 (GIb subtype), SDZB (GIIb subtype), SDLY (GIIb subtype), SDJNing (GIIc subtype), and SDQD (GIIc subtype) strains at an infection dose of 0.1 MOI. qPCR showed that transfection with siNsp9-59 significantly inhibited the expression of PEDV Nsp9. Figure 4 (A) Western blot and plaque formation assays revealed that transfection with siNsp9-59 significantly inhibited PEDV replication and infection, and exhibited broad-spectrum anti-PEDV effects, inhibiting the replication and infection of G1b, G12b, and G12c subtype PEDV strains. Figure 4 (B and C)

[0059] 3.3 Effects of siNsp9-59 transfection on cell viability

[0060] The effects of siNsp9-59 and siNC on cytotoxicity were assessed using the CCK8 assay 24 and 48 h after transfection. The results showed that cell viability was not significantly different between siNsp9-59 and siNC transfection, indicating that siNsp9-59 was not cytotoxic. Figure 5 ).

[0061] 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. A siRNA for combating porcine epidemic diarrhea virus, characterized in that, The nucleotide sequence of the positive strand of the siRNA is: GCAUAGUUGGAGAAGGUAATT; The antisense strand nucleotide sequence of the siRNA is: UUACCUUCUCCAACUAUGCTT; The siRNA targets non-structural protein 9 of porcine epidemic diarrhea virus; The siRNA can inhibit the replication of at least two porcine epidemic diarrhea virus strains from the G1b, G2b, or G2c subtypes.

2. The use of the siRNA as described in claim 1 in the preparation of a porcine epidemic diarrhea virus inhibitor.

3. A porcine epidemic diarrhea virus inhibitor, characterized in that, The inhibitor includes the siRNA as described in claim 1.

4. The use of the siRNA as described in claim 1 in the preparation of a medicament for treating diseases caused by porcine epidemic diarrhea virus infection.

5. A drug for treating diseases caused by porcine epidemic diarrhea virus infection, characterized in that, The drug comprises the siRNA as described in claim 1.

6. The drug as described in claim 5, characterized in that, The dosage form of the drug is capsule, tablet, granule, emulsion, pill, or oral liquid.

7. The drug as described in claim 5, characterized in that, The drug also includes pharmaceutically acceptable excipients.

8. The drug as described in claim 7, characterized in that, The excipients include binders, disintegrants, fillers, stabilizers, preservatives, coating materials, and / or fragrances.

9. The use of a host cell comprising the siRNA of claim 1 in the preparation of a vaccine for the prevention of porcine epidemic diarrhea virus disease.

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