Application of SQSTM1 antiviral polypeptide in preparation of product for treating porcine reproductive and respiratory syndrome

Abstract

The invention discloses application of SQSTM1 antiviral polypeptide in preparation of a product for treating porcine reproductive and respiratory syndrome, and belongs to the technical field of biological medicine. Virus infection tests find that the overexpression of the SQSTM1 gene can significantly inhibit PRRSV replication, and on the contrary, the knock-down of the gene significantly promotes virus replication. The invention proves that the SQSTM1 protein can degrade three proteins, namely GP4, GP5 and NSP12 of PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) and interacts with the three proteins; subsequently, the key amino acid region of the SQSTM1 protein for resisting PRRSV is proved, and the key amino acid region is synthesized into polypeptide. The invention proves that the SQSTM1 antiviral polypeptide has good antiviral activity and has no strain dependence, so that the SQSTM1 antiviral polypeptide has important clinical significance as a medicine for preventing and treating porcine reproductive and respiratory syndrome.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of SQSTM1 antiviral peptide in the preparation of products for treating porcine reproductive and respiratory syndrome. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV). It is characterized by reproductive disorders in pregnant sows and respiratory symptoms in pigs of all ages. It is a major pathogen threatening the global pig industry, and to date, there is still no effective eradication method. It is estimated to cause nearly $600 million in economic losses annually in the United States. The four PRRS outbreaks between 2014 and 2017 resulted in an average loss of approximately $1424.37 per sow.

[0003] Vaccination remains a key measure for the prevention and control of PRRSV in most pig farms. Clinically, vaccines include two types: live attenuated vaccines and inactivated vaccines. However, live attenuated vaccines only provide protection against homologous strains, offering poor protection against heterologous strains, and carry the risk of genetic recombination between wild-type and vaccine strains. Furthermore, the misuse of vaccines in different regions has led to numerous biosafety issues. Inactivated vaccines cannot induce sufficient neutralizing antibodies and therefore cannot provide effective protection; to date, there is still no truly effective control measure.

[0004] Therefore, the development of novel, safe, and highly effective anti-PRRSV drugs is of great significance. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the SQSTM1 antiviral peptide in the preparation of products for treating porcine reproductive and respiratory syndrome (PRRS), thereby addressing the problems existing in the prior art. This product can significantly inhibit viral replication and can be developed into a drug for the prevention and treatment of PRRS.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] In the first aspect, the present invention provides the application of overexpression of the SQSTM1 gene in the preparation of products for treating porcine reproductive and respiratory syndrome.

[0008] Preferably, the amino acid sequence of the SQSTM1 gene is shown in SEQ ID NO.3.

[0009] Preferably, the method of application includes the steps of inserting the SQSTM1 gene into a backbone vector to construct an expression vector overexpressing the SQSTM1 gene, and then transfecting it into cells.

[0010] Secondly, the present invention also provides the application of SQSTM1 antiviral peptide in the preparation of products for treating porcine reproductive and respiratory syndrome, wherein the SQSTM1 antiviral peptide includes at least one of peptide 1 and peptide 2.

[0011] Preferably, the amino acid sequence of polypeptide 1 is shown in SEQ ID NO.1; and the amino acid sequence of polypeptide 2 is shown in SEQ ID NO.2.

[0012] Preferably, the product includes a drug.

[0013] Thirdly, the present invention also provides a product for treating porcine reproductive and respiratory syndrome, the product comprising the SQSTM1 gene or the SQSTM1 antiviral peptide.

[0014] Preferably, the SQSTM1 antiviral peptide includes at least one of peptide 1 and peptide 2.

[0015] Preferably, the amino acid sequence of polypeptide 1 is shown in SEQ ID NO.1; the amino acid sequence of polypeptide 2 is shown in SEQ ID NO.2; and the amino acid sequence of the SQSTM1 gene is shown in SEQ ID NO.3.

[0016] Fourthly, the present invention also provides the use of the product in the preparation of a drug for treating porcine reproductive and respiratory syndrome.

[0017] The present invention discloses the following technical effects:

[0018] This study found that the SQSTM1 gene was significantly upregulated in the lungs of pigs infected with HP-PRRSV, and that the SQSTM1 gene was also significantly upregulated after HP-PRRSV infection of susceptible MARC-145 cells. These results suggest that the SQSTM1 gene plays an important role in the PRRSV infection process.

[0019] Overexpression of the SQSTM1 gene in MARC-145 cells followed by HP-PRRSV infection. Results showed that overexpression of the SQSTM1 gene significantly inhibited PRRSV replication, PRRSVN protein expression was downregulated in a dose-dependent manner, and progeny virus titers decreased by 2 log.

[0020] Further research revealed that overexpression of the SQSTM1 gene significantly degrades multiple PRRSV proteins, including GP4, GP5, and NSP12, in a dose-dependent manner. The porcine SQSTM1 protein can also interact with PRRSV GP4, GP5, and NSP12 proteins.

[0021] In summary, the porcine SQSTM1 protein can significantly degrade multiple PRRSV proteins, including GP4, GP5, and NSP12, and can interact with these proteins. The porcine SQSTM1 antiviral peptide has significant inhibitory activity against PRRSV infection of PAMs and can be developed into a drug for the prevention and treatment of PRRS, providing a new strategy for the clinical prevention and control of PRRS and having important guiding significance for healthy pig farming. 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 The effect of SQSTM1 overexpression on PRRSV replication; where A is the expression of PRRSVN protein detected by Western Blot, and B is TCID. 50 Detect the progeny virus titer;

[0024] Figure 2 To knock down the effect of SQSTM1 on PRRSV replication; where A is the expression of PRRSVN protein detected by Western Blot, and B is TCID. 50 Detect the progeny virus titer;

[0025] Figure 3 Western blot was used to detect the degradation of PRRSVNSP12 (A), GP4 (B), and GP5 (C) proteins by SQSTM1 protein;

[0026] Figure 4 To verify the interaction between SQSTM1 protein and PRRSVNSP12(A), GP4(B) and GP5(C) proteins in the immunoprecipitation assay;

[0027] Figure 5 This study measured the key regions of the SQSTM1 protein against PRRSV; where A represents the expression of PRRSV N protein detected by Western Blot; and B represents TCID. 50 Detect the progeny virus titer;

[0028] Figure 6 The assay is for the antiviral activity of SQSTM1 antiviral peptides; where A represents peptide 1; B represents peptide 2; and C represents peptide 1 + peptide 2.

[0029] Figure 7The antiviral activity of the SQSTM1 antiviral peptide against different PRRSV strains was measured. In this study, A represents the expression of PRRSVN protein detected by Western blotting; B represents TCID... 50 Detect the progeny virus titer. Detailed Implementation

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] This invention revealed that the SQSTM1 gene was significantly upregulated after PRRSV infection using transcriptome data, and further investigated the effect of the SQSTM1 gene on viral replication and its molecular mechanism. Experimental results showed that overexpression of SQSTM1 significantly inhibited PRRSV replication. Further investigation into the antiviral mechanism revealed that SQSTM1 can degrade multiple PRRSV proteins, including GP4, GP5, and NSP12, and can interact with these viral proteins individually. Finally, the key amino acid regions of SQSTM1 for antiviral activity were identified, and an antiviral peptide was synthesized from these key regions. This antiviral peptide significantly inhibited PRRSV replication in a dose-dependent manner; the SQSTM1 antiviral peptide also exhibited antiviral activity against different PRRSV strains.

[0036] Sources of materials used in the examples:

[0037] MARC-145 cells were preserved in our laboratory; porcine alveolar macrophages (PAMs) were derived from 8-week-old healthy pigs; antiviral peptides were synthesized by Shanghai GenScript Biotech Co., Ltd.; siRNA was synthesized by Geneplus Biotech Ltd.; Q5 premixed high-fidelity enzyme, EcoRI, XhoI, and T4 DNA ligase were all purchased from NEB; mouse-derived HA, Myc, and α-tubulin antibodies were all purchased from ProteinTech Biotech Ltd.; mouse-derived anti-PRRSVN protein monoclonal antibody was prepared in our laboratory; HRP-labeled goat anti-mouse secondary antibody was purchased from TransGen Biotech Ltd.; ECL kit was purchased from Beyotime Biotech Ltd.; HP-PRRSV strain (GD-HD, GenBank ID: KP793736), LP-PRRSV strain (VR2332, GenBank ID: EF442771), and NADC30-Like strain (GenBank ID: KX766379) were used.

[0038] The amino acid sequence of polypeptide 1 (SEQ ID NO.1): EVEGTAQ;

[0039] The amino acid sequence of peptide 2 (SEQ ID NO.2): SKEVDPSTGELQSLQMPESEGPSSLNPSQEGPTGLKEAALYPHLPPEADP;

[0040] The amino acid sequence of the protein encoded by the SQSTM1 gene (SEQ ID NO.3): MRGGASPAFPTKAARPLPLPP PPPLPPPGSSLAPQLVVMASLTVKAYLLGKEDAAREIRRFSFCFSPEPEAEAEAAAGPGPCERLLSRVAALFPVLRPGGFQAHYRDEDGDLVAFSSDEELTMAMSYVKDDIFRIYIKEKKECRRDHRPPCAQEVPRNMVHPNVICDGCNGPVVGTRYKCSVCPDYDLCAACEGKGIHKEHSKLAFPSPFGHLSEGFSHSRWLRKLRHGHFGWPGWEMGPPGNWSPRPPR AGDARPGPAEESASGPSEDPNVNFLKNVGESVAAALSPLGIEVDIDVEHGGKRSRLTPTSPGSSSTEDKCSSQPSSCSSDPSKPDGEVEGTAQCLVEQMNKIALESGVQPEEQM ESDNCSGGDDDWTHLSSKEVDPSTGELQSLQMPESEGPSSLNPSQEGPTGLKEAALYPHLPPEADPRLIESLSQMLSMGFSDEGGWLTRLLQTKNYDIGAALDTIQYSKHPPPL.

[0041] Example 1: Construction of porcine SQSTM1 eukaryotic expression plasmid

[0042] Take 10 6 Total RNA was extracted from porcine alveolar macrophages (PAMs) using the TRIzol method, reverse transcribed into cDNA, and used as a template for PCR amplification of the SQSTM1 gene. Then, the target gene and the pCMV-Myc vector were double-digested with EcoRI and XhoI, respectively. The digested target gene was cloned into the pCMV-Myc eukaryotic expression vector to construct the pCMV-Myc-SQSTM1 recombinant expression plasmid. Sequencing was performed, and plasmids with correct sequencing results were used for subsequent experiments.

[0043] PCR amplification system: 25 μL Q5 premixed high-fidelity enzyme, 1 μL cDNA template, 2.5 μL upstream primer, 2.5 μL downstream primer, 19 μL ddH2O, totaling 50 μL. PCR amplification program: 98℃ for 10 s, 55℃ for 15 s, 72℃ for 30 s, for a total of 30 cycles.

[0044] Upstream primer sequence: CCGGAATTCATGGCGTCGCTCACCGTGAAGGCCTACCTG (SEQ ID NO.4);

[0045] Downstream primer sequence: CCGCTCGAGTCACAATGGCGGGGAGTGCTTTGAA (SEQ ID NO.5).

[0046] Note: Italics in the primer sequence represent enzyme cleavage sites.

[0047] Double enzyme digestion-ligation system: 1 μL T4 DNA ligase, 2 μL 10× Buffer, 50 ng pCMV-Myc vector, 30 ng target gene, total 20 μL.

[0048] Example 2: Overexpression of SQSTM1 significantly inhibited PRRSV replication

[0049] MARC-145 cells in good growth condition were seeded in 24-well plates and cultured in a cell culture incubator for 18-24 hours until confluence reached 70%. Different doses (0, 125, 250, 500 ng) of pCMV-Myc-SQSTM1 plasmid were transfected into MARC-145 cells, and the total transfection plasmid was supplemented to 500 ng with empty pCMV-Myc vector. 12 hours after transfection, the cells were infected with 0.1% MOIPRRSV. 36 hours after viral infection, cell culture supernatant and cells were collected. Western blot analysis was performed to detect PRRSVN protein expression in each experimental group, and TCID values ​​were measured. 50 Detect the viral titer of the supernatant neutron generation.

[0050] Results: MARC-145 cells were transfected with different doses (0, 125, 250, 500 ng) of pCMV-Myc-SQSTM1 plasmid. Twelve hours after transfection, the cells were infected with PRRSV. Thirty-six hours post-infection, cell culture supernatant and cells were collected for progeny virus titer and protein assays. Compared with the control group, overexpression of SQSTM1 (500 ng) resulted in lower TCID values. 50 The expression of the porcine SQSTM1 gene significantly inhibited PRRSV replication, with a decrease of 2 log. Furthermore, Western blotting analysis showed a significant decrease in PRRSVN protein levels. These experimental results indicate that overexpression of the porcine SQSTM1 gene can significantly suppress PRRSV replication. Figure 1 (AB).

[0051] Example 3: Knocking down SQSTM1 significantly promotes PRRSV replication

[0052] MARC-145 cells in good growth condition were seeded into 12-well plates and cultured in a cell culture incubator for 18-24 hours until confluence reached 70%. MARC-145 cells were transfected with 100 nM siNC and different concentrations (25, 50, 100 nM) of siSQSTM1. Twelve hours after transfection, the cells were infected with 0.1% MOIPRRSV. Thirty-six hours after viral infection, cell culture supernatant and cells were collected. Western blot analysis was performed to detect PRRSV N protein expression in each experimental group, and TCID values ​​were also analyzed. 50 Detect the viral titer of the supernatant neutron generation.

[0053] siNC sequence: TCAACTTAATCCTGCTA (SEQ ID NO. 6);

[0054] siSQSTM1 sequence: CATCCAGTATTCAAAGCAT (SEQ ID NO.7).

[0055] Results: Contrary to the results of SQSTM1 gene overexpression, SQSTM1 gene knockdown significantly promoted PRRSV replication. Figure 2 (AB).

[0056] Example 4: SQSTM1 degrades multiple proteins of PRRSV GP4, GP5, and NSP12.

[0057] MARC-145 cells in good growth condition were seeded in 24-well plates and cultured for 18-24 hours until confluence reached 70%. Different doses (0, 250, 375, 500 ng) of pCMV-Myc-SQSTM1 plasmid were co-transfected into MARC-145 cells with 500 ng of different PRRSV gene eukaryotic expression vectors pCAGGS-HA-GP4, pCAGGS-HA-GP5, and pCAGGS-HA-Nsp12. Cell samples were collected 36 hours after transfection. Western blot analysis was performed to detect the expression of GP4, GP5, and Nsp12 proteins. The construction methods for pCAGGS-HA-GP4, pCAGGS-HA-GP5, and pCAGGS-HA-Nsp12 eukaryotic expression vectors are the same as in Example 1 (RNA was extracted from PRRSV viral fluid and reverse transcribed into cDNA. Using cDNA as a template, GP4, GP5, and Nsp12 gene fragments were amplified by PCR. Using pCAGGS-HA as a vector, the target gene and pCAGGS-HA vector were digested with Kpn I and Nhe I, respectively. The digested target gene was cloned into the pCAGGS-HA vector to construct the pCAGGS-HA-GP4, pCAGGS-HA-GP5, and pCAGGS-HA-Nsp12 recombinant expression vectors).

[0058] PCR amplification system: 25 μL Q5 premixed high-fidelity enzyme, 1 μL cDNA template, 2.5 μL upstream primer, 2.5 μL downstream primer, 19 μL ddH2O, totaling 50 μL. PCR amplification program: 98℃ for 10 s, 55℃ for 15 s, 72℃ for 30 s, for a total of 30 cycles.

[0059] GP4 upstream primer sequence: CGGGGTACCATGGCTGCGCCCTTTCTTTTCCTC (SEQ ID NO.8);

[0060] GP4 downstream primer sequence: CTAGCTAGCTCAAATTGCCAGTAGGATGGCAAA (SEQ ID NO.9).

[0061] GP5 upstream primer sequence: CGGGGTACCATGTTGGGGAAGTGCTTGACCGC (SEQ ID NO.10);

[0062] GP5 downstream primer sequence: CTAGCTAGCCTAGAGACGACCCCATTGTTCCGC (SEQ ID NO.11).

[0063] Nsp12 upstream primer sequence: CGGGGTACCATGGGCCGCCATTTTACCTGGT (SEQ ID NO.12);

[0064] Nsp12 downstream primer sequence: CTAGCTAGCTTAATTCAGGCCTAAAGTTGGTTC (SEQ ID NO.13).

[0065] Note: Italics in the primer sequence represent enzyme cleavage sites.

[0066] Double enzyme digestion-ligation system: 1 μL T4 DNA ligase, 2 μL 10× Buffer, 50 ng pCMV-Myc vector, 30 ng target gene, total 20 μL.

[0067] Results: To explore the antiviral mechanism, porcine SQSTM1 gene was co-transfected with various PRRSV proteins into MARC-145 cells. Western blot analysis of protein expression revealed that SQSTM1 could degrade multiple PRRSV proteins, including GP4, GP5, and NSP12, in a dose-dependent manner. Figure 3 (AC).

[0068] Example 5: SQSTM1 interacts with PRRSV GP4, GP5 and NSP12 proteins, respectively.

[0069] MARC-145 cells in good growth condition were seeded in 6-well plates and cultured in a cell culture incubator for 18-24 h until confluence reached 70%. The pCMV-Myc-SQSTM1 plasmid was co-transfected into MARC-145 cells with 1.0 μg pCAGGS-HA-GP4, 1.0 μg pCAGGS-HA-GP5, and 1.0 μg pCAGGS-HA-Nsp12 plasmids, respectively. Cell samples were collected 36 h post-transfection. NP40 cell lysis buffer containing PMSF protease inhibitor was added, and the cells were lysed on ice for 30 min. After centrifugation at 12000g for 15 min, the supernatant was collected into a new 1.5 mL EP tube and kept on ice for later use. Take 100 μL of Protein G Megbeads, add 2 μg of mouse HA antibody, incubate at 37°C for 1 h, wash twice with PBS; then add the cell lysis supernatant, incubate overnight with gentle shaking at 4°C; after brief centrifugation, collect the bead, add an equal volume of 2× Loading buffer, boil in water for 5 min; after brief centrifugation, take the supernatant for Western Blot detection.

[0070] Results: Immunoprecipitation experiments revealed that SQSTM1 can interact with PRRSV GP4, GP5, and NSP12, respectively. Figure 4 (AC).

[0071] Example 6: Determination of Key Antiviral Regions of SQSTM1

[0072] Construction of SQSTM1 protein truncated eukaryotic expression vectors: Using pCMV-Myc-SQSTM1 plasmid as a template, SQSTM1-ΔPB1, SQSTM1-ΔZZ, SQSTM1-ΔLIR, and SQSTM1-ΔUBA mutant gene fragments were amplified respectively. Then, they were cloned into the pCMV-Myc eukaryotic expression vector using EcoRI and KpnI restriction sites to construct pCMV-Myc-SQSTM1-ΔPB1, pCMV-Myc-SQSTM1-ΔZZ, pCMV-Myc-SQSTM1-ΔLIR, and pCMV-Myc-SQSTM1-ΔUBA recombinant plasmids. The specific construction method is the same as in Example 1.

[0073] PCR amplification system: 25 μL Q5 premixed high-fidelity enzyme, 1 μL cDNA template, 2.5 μL upstream primer, 2.5 μL downstream primer, 19 μL ddH2O, totaling 50 μL. PCR amplification program: 98℃ for 10 s, 55℃ for 15 s, 72℃ for 30 s, for a total of 30 cycles.

[0074] SQSTM1-ΔPB1 upstream primer sequence:

[0075] SQSTM1-ΔPB1 downstream primer sequence:

[0076] SQSTM1-ΔZZ upstream primer sequence:

[0077] SQSTM1-ΔZZ downstream primer sequence:

[0078] SQSTM1-ΔLIR upstream primer sequence:

[0079] SQSTM1-ΔLIR downstream primer sequence:

[0080] SQSTM1-ΔUBA upstream primer sequence:

[0081] SQSTM1-ΔUBA downstream primer sequence:

[0082] Note: In primer sequences, italics and underlines represent enzyme cleavage sites.

[0083] Double enzyme digestion-ligation system: 1 μL T4 DNA ligase, 2 μL 10× Buffer, 50 ng pCMV-Myc vector, 30 ng target gene, total 20 μL.

[0084] Determination of key antiviral regions of SQSTM1: MARC-145 cells in good growth condition were seeded in 12-well plates and cultured for 18-24 hours until 70% confluence was achieved. MARC-145 cells were transfected with 1.0 μg of recombinant plasmids pCMV-Myc-SQSTM1-ΔPB1, pCMV-Myc-SQSTM1-ΔZZ, pCMV-Myc-SQSTM1-ΔLIR, and pCMV-Myc-SQSTM1-ΔUBA. 24 hours post-transfection, cells were infected with 0.1% MOIPRRSV. 36 hours post-infection, culture supernatant and cells were collected. Western blot analysis was performed to detect PRRSVN protein expression in each experimental group, and TCID values ​​were measured. 50 The viral titer in the supernatant neutrons was detected. Empty vector and pCMV-SQSTM1-Myc plasmid were used as controls.

[0085] Results: The key amino acid regions of SQSTM1 for antiviral activity were identified. The effects of recombinant plasmids with deletions in these key regions (pCMV-Myc-SQSTM1-ΔPB1, pCMV-Myc-SQSTM1-ΔZZ, pCMV-Myc-SQSTM1-ΔLIR, and pCMV-Myc-SQSTM1-ΔUBA) on viral replication were verified. Western Blot and TCID 50 The results showed that the LIR region and the UBA region were the main antiviral regions. Figure 5 (AB).

[0086] Example 7: Antiviral activity of SQSTM1 antiviral peptide

[0087] The key antiviral regions of SQSTM1 identified in Example 6 were synthesized into peptides, named peptide 1 (LIR region) and peptide 2 (UBA region), respectively. PAMs were seeded onto 24-well cell culture plates and cultured for 4 hours. After complete cell adhesion, different amounts (0, 20, 40, 80, 160, 320, 640, 1280 μg) of peptide 1, peptide 2, and SQSTM1 antiviral peptide (peptide 1 to peptide 2 mass ratio 1:1) were added to pretreat the cells for 1 hour. Simultaneously, a control group was prepared by adding 1280 μg of a random peptide with a scrambled amino acid sequence (sequence information: DPSGTQSQQHLDPNKEVAATGSSQSLYPAQQSEAQM, SEQ ID NO. 22). Cells were then infected with 0.01 MOI PRRSV. 36 hours post-infection, the cell culture supernatant was collected. TCID 50 Detect the viral titer of the supernatant neutron generation.

[0088] Results: PAMs were transfected with peptides synthesized from two key regions. Simultaneously, PAMs were transfected with peptide 1 and peptide 2 alone. One hour after transfection, cells were infected with 0.01 MOIPRRSV. Cell supernatant was collected 36 hours post-infection. The results showed that both peptide 1 and peptide 2 exhibited significant antiviral activity compared to the control group. Figure 6 (A and B), however, the combined use of the two peptides is more effective. Transfection with the SQSTM1 antiviral peptide at 160 μg significantly reduced progeny virus titers; with increasing SQSTM1 antiviral peptide concentration, the progeny virus titers gradually decreased. Figure 6 (C)

[0089] Example 8: Antiviral activity of SQSTM1 antiviral peptide against different PRRSV strains

[0090] PAMs were seeded into 24-well cell culture plates and cultured for 4 hours. Once cells were fully adherent, they were pretreated for 1 hour with different concentrations (320, 640, 1280 μg) of SQSTM1 antiviral peptides (peptide 1 and peptide 2 in a 1:1 mass ratio). Simultaneously, a control group was prepared by adding 1280 μg of a random peptide with scrambled amino acid sequences (sequence information: DPSGTQSQQHLDPNKEVAATGSSQSLYPAQQSEAQM, SEQ ID NO.22). Cells were then infected with 0.01 MOI of different PRRSV strains (GD-HD, GenBank ID: KP793736; VR2332, GenBank ID: EF442771; NADC30-Like, GenBank ID: KX766379). 36 hours post-infection, cells and culture supernatant were collected. Western blot analysis was performed to detect PRRSVN protein expression, TCID... 50 Detect the viral titer of the supernatant neutron generation.

[0091] Results: The SQSTM1 antiviral peptide exhibited antiviral activity against different PRRSV strains without strain dependence. Figure 7 (AB).

[0092] Western blot detection and TCID in all the above embodiments 50 The specific experimental procedures for detection are as follows:

[0093] Western blot: (1) Cell sample processing: Collect cells by trypsin digestion into EP tubes, add NP40 lysis buffer and incubate on ice for 30 min to allow the cells to fully lyse. Then centrifuge at 12,000g for 8 min, aspirate the supernatant into a new EP tube, add 5× Loading Buffer, and boil for 10 min. (2) SDS-PAGE: Install the protein gel plate and gel tank, add 8 μL of sample to each well, and run electrophoresis at 200V constant voltage for 40 min. (3) Transfer: After SDS-PAGE, remove the protein gel and install the transfer clamp according to the filter paper, protein gel, PVDF membrane, and filter paper. Transfer the membrane at 100V for 1 h. (4) Blocking: Remove the PVDF membrane and place it in 5% skim milk powder for 2 h at room temperature. (5) Incubation with primary antibody: Wash the membrane 3 times with PBST, dilute the mouse anti-PRRSVN antibody 1:1000, and dilute the mouse HA antibody 1:2000. Incubate at 37℃ for 1 h. (6) Incubation with secondary antibody: Wash the membrane 3 times with PBST, dilute HRP-labeled goat anti-mouse secondary antibody 1:5000, and incubate at 37℃ for 1 h. (7) Color development: ECL luminescence color development.

[0094] TCID 50 Detection: MARC-145 cells in good growth condition were seeded into 96-well plates and incubated in a cell culture incubator for 16-24 hours until cell confluence reached 80%. The cell culture supernatant after virus infection was collected and serially diluted 10-fold from 10... -1 Up to 10 -7 Eight replicates were made for each dilution. Cells were added and incubated in a cell culture incubator for 1 hour. The medium was then replaced with 3% FBS DMEM and cultured for another 5 days. Cytopathic effects were observed under a microscope, and the TCID of the virus was calculated using the Reed-Muench method. 50 .

[0095] 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. Application of SQSTM1 gene overexpression in the preparation of products for treating porcine reproductive and respiratory syndrome.

2. The application according to claim 1, characterized in that, The amino acid sequence of the SQSTM1 gene is shown in SEQ ID NO.

3.

3. The application according to claim 2, characterized in that, The method of the application includes the steps of inserting the SQSTM1 gene into a backbone vector to construct an expression vector overexpressing the SQSTM1 gene, and then transfecting it into cells.

4. The application of SQSTM1 antiviral peptide in the preparation of products for treating porcine reproductive and respiratory syndrome, characterized in that, The SQSTM1 antiviral peptide includes at least one of peptide 1 and peptide 2.

5. The application according to claim 4, characterized in that, The amino acid sequence of polypeptide 1 is shown in SEQ ID NO.1; the amino acid sequence of polypeptide 2 is shown in SEQ ID NO.

2.

6. The application according to claim 4, characterized in that, The products include pharmaceuticals.

7. A product for treating porcine reproductive and respiratory syndrome, characterized in that, The product includes the SQSTM1 gene as described in claim 1 or the SQSTM1 antiviral peptide as described in claim 4.

8. The product according to claim 7, characterized in that, The SQSTM1 antiviral peptide includes at least one of peptide 1 and peptide 2.

9. The product according to claim 8, characterized in that, The amino acid sequence of polypeptide 1 is shown in SEQ ID NO.1; the amino acid sequence of polypeptide 2 is shown in SEQ ID NO.2; and the amino acid sequence of the SQSTM1 gene is shown in SEQ ID NO.

3.

10. The use of the product according to any one of claims 7-9 in the preparation of a medicament for treating porcine reproductive and respiratory syndrome.

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