Polypeptide for resisting PEDV / PRRSV (porcine epidemic diarrhea virus / porcine reproductive and respiratory syndrome virus) proliferation, application and medicine

Abstract

The invention belongs to the field of biology, and discloses a polypeptide for resisting PEDV / PRRSV (porcine epidemic diarrhea virus / porcine reproductive and respiratory syndrome virus) proliferation, and the amino acid sequence of the polypeptide is as shown in SEQ ID NO. 1 or SEQ ID NO. 2 and as shown in SEQ ID NO. 3 or SEQ ID NO. 4. The polypeptide can significantly inhibit PEDV / PRRSV replication, and the inhibition effect shows obvious concentration dependence. Meanwhile, the invention further provides application and a medicine of the polypeptide.

Description

Technical Field

[0001] The present invention relates to the biological field, and in particular to a polypeptide for resisting PEDV / PRRSV virus proliferation, and its application and medicine. Background Art

[0002] Porcine epidemic diarrhea virus (PEDV) belongs to the alphacoronavirus genus and can infect pigs of all ages. Its clinical manifestations include vomiting, diarrhea, and dehydration. It is highly lethal to newborn piglets, with a mortality rate of up to 100%. Since 2010, the emergence of mutant strains has increased PEDV infection rates by 50.21%-62.10%, causing significant economic losses to the global pig industry. Vaccination is the primary means of preventing and controlling PEDV infection. However, PEDV primarily affects newborn piglets, causing them to become ill and die. Newborn piglets have an immature immune system and rely on passive immunity from sow colostrum, making vaccines unsatisfactory in most cases. Furthermore, PEDV strains continue to mutate, further reducing the protective efficacy of vaccines.

[0003] PRRSV (Predatory Respiratory Syndrome) is a highly contagious, immunosuppressive disease that infects pigs. It is classified as a Category B / II infectious disease by both the International Veterinary Office and my country, causing significant economic losses to the global swine industry. Vaccination remains the primary means of preventing and controlling PRRS. However, continuous mutations in the PRRSV genome, leading to antigenic shift and drift, render traditional vaccines less than ideally protective against emerging and heterologous strains. More importantly, overuse of attenuated vaccines poses safety risks such as reversion to virulence and recombination, increasing PRRSV infection rates in pigs and driving mutations, exacerbating strain diversity and creating a vicious cycle.

[0004] Among the drugs against PEDV, one type of drug is now gradually gaining attention, namely peptide drugs. Related literature can be found in:

[0005] The publication number is CN119080952A, and the subject is a patent application for a polypeptide for enhancing the replication of porcine epidemic diarrhea virus and its application. The polypeptide is a membrane-penetrating polypeptide targeting the PEDV replication stage and uses arginine 58 in the nucleocapsid N protein sequence of PEDV as the target.

[0006] The publication number is CN116913371A, and the subject is a patent application for a polypeptide that targets Nsp5 and can inhibit PEDV. The polypeptide specifically binds to PEDV Nsp5 and conducts virus inhibition experiments at the cellular level. The polypeptide concentration that effectively inhibits viral replication reaches 3.125μM, with outstanding virus inhibition effect.

[0007] For peptide drugs against PRRSV, please refer to the following literature:

[0008] Publication number CA2751655A1, the subject of the patent application is a protein encoded by the polynucleotide of porcine reproductive and respiratory syndrome virus (PRRSV). The proposal was put forward in 1998, 28 years have passed. With the continuous mutation of the virus, the polypeptide may not be suitable for the current epidemic prevention situation.

[0009] Publication number CN101495138A is a patent application for the identification and use of protective antigenic determinants against porcine reproductive and respiratory syndrome virus (PRRSV). The PAD is produced by heterodimers composed of the GP5 and M proteins of PRRSV, in which the extracellular domain of GP5 exhibits different N-glycosylation states. PAD and its encoding nucleic acid can be used to develop anti-PAD antibodies, effectively providing vaccines that protect against PRRSV infection. This proposal was proposed almost 20 years ago.

[0010] Through the analysis of existing technologies, we can clearly draw the following conclusions:

[0011] 1. There are few peptide solutions for inhibiting PRRSV viral proliferation;

[0012] 2. The relevant peptide scheme has been proposed for many years, and the technology has hardly been updated in the past three decades.

[0013] The main research direction of this application is to try to develop a new polypeptide that can effectively inhibit the proliferation of PEDV / PRRSV. Summary of the Invention

[0014] The purpose of the present invention is to provide a polypeptide for resisting PEDV / PRRSV virus proliferation, which can significantly inhibit PEDV / PRRSV replication, and the inhibitory effect shows obvious concentration dependence.

[0015] At the same time, the present invention also provides applications and medicines of the polypeptide.

[0016] To achieve the above objectives, the present application discloses a polypeptide for resisting PEDV virus proliferation, the amino acid sequence of the polypeptide being shown in SEQ ID NO.1 or SEQ ID NO.2.

[0017] The mechanism of the present invention is:

[0018] RNAi is a post-transcriptional gene silencing mechanism first observed in petunias in 1990. RNAi is mediated by Dicer, a member of the ribonuclease III (RNase III) family, which directs the Argonaute protein (AGO), an integral component of the RNA-induced silencing complex (RISC), to target cognate mRNAs. Its cleavage products include microRNAs (miRNAs) processed from hairpin precursor miRNAs and small interfering RNAs (siRNAs) processed from long dsRNAs. Several studies have established RNAi as a highly conserved antiviral immunity mechanism in eukaryotes, playing a crucial role in antiviral defenses in fungi, plants, nematodes, and insects.

[0019] In antiviral RNAi, viral double-stranded RNA (dsRNA) produced during viral replication is cleaved by the nuclease Dicer into virus-derived small interfering RNAs (vsiRNAs). These RNAs are then loaded onto Argonaute (AGO)2 to form an RNA-induced silencing complex (RISC), which cleaves complementary viral RNA to achieve antiviral effects. To counteract this, viruses encode viral suppressors of RNAi (VSRs) that target key steps in the RNAi pathway.

[0020] Therefore, if VSR can be targeted, the antiviral effect can be improved.

[0021] The present invention selects a VSR targeting peptide of the nucleocapsid protein of PEDV, which can target the VSR and exhibit an antiviral effect on PEDV.

[0022] Among the two polypeptides disclosed above, it has been verified that both PEDV-N-DRI (SEQ ID NO. 1) and PEDV-N-R8 (SEQ ID NO. 2) polypeptides can significantly inhibit PEDV replication, and the inhibitory effect shows obvious concentration dependence. In addition, at the same concentration, the anti-PEDV effect of PEDV-N-R8 polypeptide is better than that of PEDV-N-DRI polypeptide.

[0023] At the same time, the present invention also discloses the use of the polypeptide described above in the preparation of drugs for resisting PEDV virus proliferation.

[0024] At the same time, the present invention also discloses a drug for resisting PEDV virus proliferation, which contains the polypeptide as described above.

[0025] At the same time, the present invention also discloses a polypeptide for resisting the proliferation of PRRSV virus, and the amino acid sequence of the polypeptide is shown as SEQ ID NO.3 or SEQ ID NO.4.

[0026] The mechanism of the present invention is substantially the same as that of the aforementioned polypeptides that inhibit PEDV virus proliferation.

[0027] At the same time, the present invention also discloses the use of the polypeptide described above in preparing a drug for resisting PRRSV virus proliferation.

[0028] Finally, the present invention also discloses a drug for resisting PRRSV virus proliferation, which contains the polypeptide described above.

[0029] The present invention has the following advantages and effects compared to the prior art:

[0030] The present invention selects a VSR targeting peptide of the nucleocapsid protein of PEDV / PRRSV, which can target the VSR and exhibit an antiviral effect against PEDV / PRRSV;

[0031] It has been verified that both PEDV-N-DRI (SEQ ID NO. 1) and PEDV-N-R8 (SEQ ID NO. 2) polypeptides can significantly inhibit PEDV replication, and the inhibitory effect shows obvious concentration dependence. In addition, at the same concentration, the anti-PEDV effect of PEDV-N-R8 polypeptide is better than that of PEDV-N-DRI polypeptide.

[0032] Both PRRSV-N-DRI (SEQ ID NO. 1) and PRRSV-N-R8 (SEQ ID NO. 2) polypeptides can significantly inhibit PRRSV replication, and the inhibitory effect shows obvious concentration dependence. In addition, at the same concentration, the anti-PRRSV effect of PRRSV-N-R8 polypeptide is better than that of PRRSV-N-DRI polypeptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The results of the cytotoxicity experiment of PEDV-N-DRI polypeptide on IPEC-J2 cells are shown;

[0034] Figure 2 The results of the cytotoxicity experiment of PEDV-N-R8 peptide on IPEC-J2 cells are shown;

[0035] Figure 3 This is the result of the inhibition of PEDV proliferation by PEDV-N-DRI polypeptide;

[0036] Figure 4 This is the result of the inhibition of PEDV proliferation by PEDV-N-R8 polypeptide;

[0037] Figure 5 The results of the cytotoxicity experiment of PRRSV-N-DRI polypeptide on IPEC-J2 cells are shown;

[0038] Figure 6The results of the cytotoxicity test of PRRSV-N-R8 polypeptide on IPEC-J2 cells are shown;

[0039] Figure 7 This is the result of the PRRSV-N-DRI polypeptide inhibiting the proliferation of PRRSV;

[0040] Figure 8 This is the result of the inhibition of PRRSV-N-R8 polypeptide on the proliferation of PRRSV. DETAILED DESCRIPTION

[0041] The present invention will be described clearly and completely below in conjunction with the examples of the present invention. In the description of the present invention, it should be noted that, where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer were used. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially.

[0042] Example 1 Design of polypeptide

[0043] Through functional analysis of PEDV structural and non-structural proteins, it was found that the nucleocapsid protein (N protein) can act as a viral VSR to block the cleavage of the host Dicer.

[0044] Based on this, a variety of software were used to compare and analyze the PEDV N protein sequence as well as the two-dimensional and three-dimensional structures.

[0045] The N protein can self-assemble into homodimers. Based on the N protein homodimer structure, its binding site to viral RNA, and sequence variation in the corresponding region, the VTP polypeptide sequence was designed. The peptides were then synthesized by a biotechnology company, resulting in two peptides, named PEDV-N-DRI (FLLAAVNPALSAIQAYGPPRRRQRRKKRGY, SEQ ID NO. 1) and PEDV-N-R8 (RRRRRRRRGYAQIASLAPNVAALLF, SEQ ID NO. 2).

[0046] Example 2 Detection of Anti-PEDV Activity of Polypeptides

[0047] (1) The polypeptide synthesized in Example 1 was added to phosphate buffer (PBS: 135 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8 mM K2HPO4, pH 7.2) to prepare a 1 mM solution, filtered through a 0.22 μM microporous filter for sterilization, and stored at -80°C after aliquoting;

[0048] (2) Cytotoxicity test of peptides: The dissolved peptides were diluted with serum-free DMEM to obtain dilutions with concentrations of 50uM, 5uM, 0.5uM, and 0.05uM. IPEC-J2 cells and Marc-145 cells were used, respectively, and the cells were seeded in 96-well plates. After the cells were aggregated, the culture medium was discarded and the cells were washed 3 times with PBS. Different dilution concentrations of PEDV-N-DRI and PEDV-N-R8 peptides were added to the 96-well plates of IPEC-J2 cells, and 8 wells were inoculated for each dilution concentration. At the same time, a normal cell control group and a blank control group were set up. The final volume of each well was 100uL. The cells were placed in a 37°C, 5% CO2 incubator and cultured for 48 hours. Finally, the OD value of each well was measured by CCK8 method to calculate the cell survival rate.

[0049] The results are as follows Figure 1 and Figure 2 As shown, at a concentration of 50 uM, both PEDV-N-DRI and PEDV-N-R8 polypeptides were non-toxic to IPEC-J2 cells and could be used for subsequent experiments.

[0050] Example 3 Inhibitory Effects of PEDV-N-DRI and PEDV-N-R8 Peptides on PEDV Proliferation

[0051] IPEC-J2 cells were seeded in 12-well plates. After confluence, the culture medium was discarded and the cells were washed three times with PBS. PEDV-N-DRI and PEDV-N-R8 peptides were added to the cells at concentrations of 50 μM, 5 μM, 0.5 μM, and 0.05 μM, respectively. IPEC-J2 cells were infected with PEDV virus at an MOI of 0.1. One hour after infection, the supernatant was discarded, the cells were washed three times with PBS, and DMEM medium containing 2.5 μg / mL EDTA-free trypsin and PEDV-N-DRI and PEDV-N-R8 peptides were added. The peptide concentration in each well remained the same as that at the time of infection.

[0052] At the same time, a non-infected control group and a PEDV-infected control group were set up.

[0053] The cells were incubated at 37°C, 5% CO2 for 24 hours. The cytopathic effects were observed under a microscope, and the viral titer in each group of cells was measured to evaluate the inhibitory effect of the peptide on PEDV proliferation. The experiment was repeated three times.

[0054] The cells obtained in the above steps were frozen and thawed three times, centrifuged at 10,000 rpm and 4°C for 10 minutes, and the supernatant was collected. Healthy Vero cells were evenly plated in a 96-well plate and the virus titer was determined after the cells were confluent.

[0055] The virus solution was diluted 10 times in a row using serum-free DMEM medium containing 2.5 μg / mL EDTA-free trypsin.-1 ~10 -10 . Aspirate the culture medium in the 96-well plate and rinse the cells 2-3 times with PBS. Inoculate the diluted virus solution into a 96-well plate, inoculate each dilution gradient into 8 wells in a vertical column, inoculate 100 μL into each well, and add 100 μL DMEM culture medium to each well in the last two rows as a control. Continue to culture the cells in a 37°C, 5% CO2 incubator. After about 3-5 days, observe and record the number of cytopathic effect (CPE) wells. The TCID50 of the virus can be calculated according to the Reed-Muench method.

[0056] The results are as follows Figure 3 and Figure 4 As shown in the results, both PEDV-N-DRI and PEDV-N-R8 polypeptides can significantly inhibit PEDV replication, and the inhibitory effect shows obvious concentration dependence. In addition, at the same concentration, the anti-PEDV effect of PEDV-N-R8 polypeptide is better than that of PEDV-N-DRI polypeptide.

[0057] Example 4: Design of polypeptides

[0058] Through functional analysis of PRRSV structural and non-structural proteins, it was found that the nucleocapsid protein (N protein) can act as a viral VSR to block the cleavage of the host Dicer.

[0059] Based on this, a variety of software were used to compare and analyze the PRRSV N protein sequence as well as the two-dimensional and three-dimensional structures.

[0060] The N protein can self-assemble into homodimers. Based on the N protein homodimer structure, its binding sites to viral RNA, and sequence variation in the corresponding regions, the VTP polypeptide sequence was designed. The peptides were then synthesized by a biotechnology company, resulting in two peptides, named PRRSV-N-DRI (QNFATQISSLCLQREPPRRRQRRKKRGY, SEQ ID NO. 3) and PRRSV-N-R8 (RRRRRRRRERQLCLSSIQTAFNQ, SEQ ID NO. 4).

[0061] Example 5 Detection of Anti-PRRSV Activity of Polypeptides

[0062] (1) The polypeptide synthesized in Example 4 was added to phosphate buffer (PBS: 135 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8 mM K2HPO4, pH 7.2) to prepare a 1 mM solution, filtered through a 0.22 μM microporous filter for sterilization, and stored at -80°C after aliquoting;

[0063] (2) Cytotoxicity test of polypeptides: The dissolved polypeptides were diluted with serum-free DMEM to obtain dilutions with concentrations of 50uM, 5uM, 0.5uM and 0.05uM. Marc-145 cells were used and inoculated into 96-well plates. After the cells were aggregated, the culture medium was discarded and the cells were washed 3 times with PBS. Different dilution concentrations of PRRSV-N-DRI and PRRSV-N-R8 polypeptides were added to 96-well plates of Marc-145 cells. Eight wells were inoculated for each dilution concentration. A normal cell control group and a blank control group were set up at the same time. The final volume of each well was 100uL. The cells were placed in a 37°C, 5% CO2 incubator and cultured for 48 hours. Finally, the OD value of each well was measured using the CCK8 method to calculate the cell survival rate.

[0064] The results are as follows Figure 5 and Figure 6 As shown, at a concentration of 50 uM, both PRRSV-N-DRI and PRRSV-N-R8 polypeptides were non-toxic to Marc-145 cells and could be used in subsequent experiments.

[0065] Example 6 Inhibitory Effects of Marc-145N-DRI and Marc-145-N-R8 Peptides on Marc-145 Proliferation

[0066] Marc-145 cells were seeded in 12-well plates. After confluency, the culture medium was discarded and the cells were washed three times with PBS. PRRSV-N-DRI and PRRSV-N-R8 peptides were added to the cells at concentrations of 50 μM, 5 μM, 0.5 μM, and 0.05 μM, respectively. PRRSV virus was then infected with the Marc-145 cells at an MOI of 0.1. One hour after infection, the supernatant was discarded, the cells were washed three times with PBS, and DMEM medium supplemented with 2% serum and PRRSV-N-DRI and PRRSV-N-R8 peptides were added. The peptide concentrations in each well remained the same as those used for infection. Uninfected and PRRSV-infected control groups were also established. The cells were incubated at 37°C, 5% CO₂ in a humidified incubator for 24 hours. Cytopathic effects were observed microscopically, and the inhibitory effect of the peptides on PRRSV proliferation was assessed by indirect immunofluorescence. The experiment was repeated three times.

[0067] The cells obtained in the above steps were washed 3 times with PBS, then fixed with 0.5 mL of 4% paraformaldehyde at room temperature for 15 minutes, and washed 3 times with PBS; treated with 0.1% Triton-100 at room temperature for 15 minutes, and washed 3 times with PBS. Blocked with 5% BSA at room temperature for 1 hour or at 4°C overnight, washed 3 times with PBS, and incubated with a porcine anti-PRRSV polyclonal antibody for primary antibody, incubated at 37°C for 1 hour or at 4°C overnight. Washed 3 times with PBS, and stained with Alexa Fluor 500 antibody. The cells were incubated with 488-labeled goat anti-swine secondary antibody at 37°C for 1 h. After washing three times with PBS, the cell fluorescence was observed under a fluorescence microscope.

[0068] The results are as follows Figure 7 and Figure 8 As shown, both PRRSV-N-DRI and PRRSV-N-R8 polypeptides can significantly inhibit PRRSV replication, and the inhibitory effect shows obvious concentration dependence. In addition, at the same concentration, the anti-PRRSV effect of PRRSV-N-R8 polypeptide is better than that of PRRSV-N-DRI polypeptide.

[0069] Summarize:

[0070] 1. The above experiments show that the polypeptide of the present invention is non-cytotoxic, safe and reliable, and can be used as an anti-PEDV / PRRSV drug;

[0071] 2. The above experiments show that the research and development idea of ​​the VSR targeting peptide based on the nucleocapsid protein of PEDV / PRRSV of the present invention is completely feasible. It can show the inhibitory effect on PEDV / PRRSV at extremely low concentrations and is an excellent anti-PEDV / PRRSV drug.

[0072] 3. In several screenings, two peptides were selected for each virus, and it was confirmed that the PEDV-N-R8 peptide had the best anti-PEDV effect, and the PRRSV-N-R8 peptide had the best anti-PRRSV effect.

Claims

1. A polypeptide for resisting PEDV virus proliferation, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. Use of the polypeptide according to claim 1 in the preparation of a drug for inhibiting PEDV virus proliferation.

3. A drug for preventing PEDV virus proliferation, characterized in that: Contains the polypeptide according to claim 1.

4. A polypeptide for resisting PRRSV virus proliferation, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO.3 or SEQ ID NO.

4.

5. Use of the polypeptide according to claim 4 in the preparation of a drug for resisting PRRSV virus proliferation.

6. A drug for resisting PRRSV virus proliferation, characterized in that: Contains the polypeptide according to claim 4.

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