Polypeptides having activity against porcine reproductive and respiratory syndrome virus and uses thereof

Abstract

The application discloses a group of polypeptides with PRRSV (Porcine Reproductive and Respiratory Syndrome virus) activity and application thereof. The amino acid sequence of the polypeptide is shown as SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO. 4. In addition, the application also provides application of the polypeptide in preparation of a PRRSV-resistant medicine. The polypeptide is designed according to PRRSV and is optimized in structure, and compared with other known anti-virus polypeptides, the polypeptide of the application shows relatively low cytotoxicity and can also play the same anti-virus effect under the condition of 25 mu g / mL concentration, indicating that the polypeptide designed in the application has higher safety and effectiveness in medicine development. The application provides a new technical means for resisting PRRSV.

Description

Technical Field

[0001] This invention relates to a group of polypeptides with anti-porcine reproductive and respiratory syndrome virus (PRRSV) activity, and also to the application of said polypeptides in anti-PRRSV infection. This invention belongs to the field of pharmaceutical technology. Background Technology

[0002] Viral infections in pigs are one of the major challenges facing the global pig farming industry. Widely prevalent porcine pathogens include pseudorabies virus (PRV), porcine epidemic diarrhea virus (PEDV), and porcine reproductive and respiratory syndrome virus (PRRSV), causing significant economic losses to the industry. Since its initial discovery in the United States in 1987, PRRSV has been prevalent globally, frequently causing fever, anorexia, and reproductive disorders in sows, as well as respiratory diseases in piglets, resulting in substantial economic losses for the world's pig farming industry. China discovered the disease in the mid-1990s and classified it as a Class II animal disease. The disease can persist in pig herds, either as a single infection or in combination with other pathogens. In particular, subsequent complications and secondary infections exacerbate clinical symptoms and increase mortality rates. Therefore, porcine reproductive and respiratory syndrome (PRRS) is currently one of the most significant viral infectious diseases hindering the development of my country's pig farming industry.

[0003] Faced with the enormous threat posed by PRRSV to the global swine industry, vaccination has become a primary means of animal disease control. Many traditional PRRSV vaccines have been approved for production and are widely used clinically, but most new vaccines are still in the research and development stage and have not yet received production approval. Various vaccines face numerous challenges in controlling PRRSV. While inactivated vaccines pose no biosafety risks, they have low levels of neutralizing antibodies, cannot generate cellular immunity, and require multiple injections. Live vaccines suffer from insufficient protection against heterologous strains, potential virulence reversion, recombination between vaccine and wild-type strains, and a lack of markers. Subunit and virus-like particle vaccines, although non-pathogenic, offer only partial protection to susceptible animals. Nucleic acid vaccines, while simple to produce, pose potential biosafety risks, such as the integration of exogenous DNA into the host genome. PRRSV primarily infects porcine alveolar macrophages (PAMs), which are characterized by high mutation and recombination rates; existing vaccines can easily become ineffective due to viral antigen mutations.

[0004] Due to the enormous economic losses caused by PRRSV globally, an increasing number of antimicrobial peptides with anti-PRRSV activity have been discovered and designed. Antimicrobial peptides are small molecule proteins with potential antibacterial, antiviral, antifungal, antitumor, and antiparasitic activities, and are widely found in animals, plants, and microorganisms [WANG J, SONG J, YANG Z, et al. Correction to Antimicrobial Peptides with High Proteolytic Resistance for Combating Gram-Negative Bacteria[J]. J Med Chem, 2019, 62(12): 5921.]. Antimicrobial peptides can inhibit viral replication by interfering with multiple stages of the viral life cycle, and their mechanisms of action are diverse, mainly including disrupting the viral envelope, preventing the virus from binding to host cells, and interacting with specific receptors on host cells [PEN G, YANG N, TENG D, et al. A Review on the Use of Antimicrobial Peptides to Combat Porcine Viruses[J]. Antibiotics (Basel), 2020, 9(11):]. In vitro experiments have shown that the small peptide Cecropin D (CD) can attenuate apoptosis induced by PRRSV infection in the later stage and inhibit the release of viral particles [LIU X, GUO C, HUANGY, et al. Inhibition of porcine reproductive and respiratory syndrome virus by Cecropin D in vitro [J]. Infect Genet Evol, 2015, 34(7-16.], and Humancathelicidin LL37 can exhibit anti-PRRSV activity in vitro by reducing viral replication [LEVAST B, HOGAND, VAN KESSEL J, et al. Synthetic Cationic Peptide IDR-1002 and HumanCathelicidin LL37 Modulate the Cell Innate Response but Differentially Impact PRRSV Replication in vitro [J]. Front Vet Sci, 2019, 6(233.], and the results of these peptide studies demonstrate the great potential of antimicrobial peptides in the development of antiviral drugs.

[0005] In response to the significant challenges posed by PRRSV's high mutation and recombination rates and the ease with which existing vaccines become ineffective, this invention designs peptides targeting PRRSV and optimizes their structure to enhance their antiviral activity, thereby killing PRRSV and preventing it from infecting pigs. Summary of the Invention

[0006] The purpose of this invention is to provide a group of polypeptides with anti-porcine reproductive and respiratory syndrome virus (PRRSV) activity and their applications.

[0007] To achieve the above objectives, the present invention employs the following technical means:

[0008] This invention designed and synthesized the peptide Protegrin-4 (P-1), whose amino acid sequence is shown in SEQ ID NO.1, and optimized its structure. We retained the aromatic amino acids in P-1 and introduced positively charged lysine residues into P-2 (SEQ ID NO.2), increasing the cationic nature of the peptide chain and facilitating interaction with negatively charged viral envelopes or cell membranes. Lysine is also a hydrophilic amino acid, which can enhance the interaction between the peptide and the cell membrane surface. Continuous positively charged arginine residues were introduced into P-3 (SEQ ID NO.3) and P-4 (SEQ ID NO.4), resulting in strong cell penetration and enhancing the peptide's membrane-penetrating ability. HPLC and CD spectroscopy results showed that the peptide had high purity and a good secondary structure. In the cell membrane environment, it could induce significant structural changes in the peptide, mainly the formation of α-helices and β-sheets. Subsequently, we used various experimental methods to detect its antiviral activity. RT-qPCR and Western blot analyses revealed that the peptides of this invention significantly inhibited viral RNA and protein expression, exhibiting the same effect even at low doses after structural optimization. Indirect immunofluorescence assays showed that the peptides of this invention had a significant direct inactivation effect on PRRSV.

[0009] Therefore, based on the above research, this invention proposes a group of polypeptides with anti-porcine reproductive and respiratory syndrome virus (PRRSV) activity, wherein the amino acid sequences of the polypeptides are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4.

[0010] Furthermore, the present invention also proposes the application of the aforementioned polypeptide in the preparation of a drug for combating porcine reproductive and respiratory syndrome virus (PRRSV).

[0011] Preferably, the porcine reproductive and respiratory syndrome virus (PRRSV) is a highly pathogenic PRRSV.

[0012] Preferably, the polypeptide inactivates PRRSV by directly contacting it.

[0013] Compared with the prior art, the beneficial effects of the present invention are:

[0014] This invention designs and synthesizes a group of peptides with anti-porcine reproductive and respiratory syndrome virus (PRRSV) activity. Compared with other known antiviral peptides, such as Cecropin D, which exhibits effective antiviral activity against PRRSV infection and replication in vitro at a concentration of 300 μg / mL, the peptides of this invention show relatively low cytotoxicity and can exert the same antiviral effect at a concentration of 25 μg / mL, indicating that the peptides designed in this invention have high safety and efficacy in drug development. Therefore, this invention provides a new technical means for anti-PRRSV treatment. Attached Figure Description

[0015] Figure 1a -d are the high-performance liquid chromatograms and mass spectra of peptides P-1 to P-4, respectively;

[0016] Figure 1e The CD values ​​are those of polypeptides P-1 to P-4 in aqueous solution;

[0017] Figure 1f CD values ​​of peptides P-1 to P-4 in 50% TFE;

[0018] Figure 2 Figure (a) shows the toxicity assay results of peptides P-1 to P-4 on MARC-145 cells, and Figure (bd) shows the results of Calcein / PI cell viability and cytotoxicity assays.

[0019] Figure 3 The results represent the detection of antiviral activity of peptides P-1 to P-4 directly.

[0020] In the figure, a represents the RT-qPCR detection of PRRSVN gene mRNA expression; b represents the Western blot results of PRRSV-N protein expression; and c represents the bar chart of the Western blot results of PRRSV-N protein expression. Detailed Implementation

[0021] The present invention is further described below. The embodiments described herein are merely exemplary and do not constitute a limitation on the scope of the invention. Those skilled in the art should understand that modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the principles and methods of the invention, but such modifications or substitutions shall all fall within the protection scope of the present invention.

[0022] Example 1: Synthesis of a polypeptide with anti-porcine reproductive and respiratory syndrome virus (PRRSV) activity and its application in combating PRRSV.

[0023] 1. Materials and Methods

[0024] 1.1 Cells and Viruses

[0025] Cells: African green monkey kidney epithelial cells (Meat Animal Research Center-145, MARC-145) were preserved in our laboratory.

[0026] The strain: High pathogenicity porcine reproductive and respiratory syndrome virus (HP-PRRSV) was preserved in this laboratory.

[0027] 1.2 Synthesis and Purification of Polypeptides

[0028] Based on the structural characteristics of P-1 and the membrane structure of PRRSV, P-1 was cationicized to design and synthesize three derivatives: P-2, P-3, and P-4. These derivatives were then synthesized using solid-phase synthesis technology and purified and analyzed by high-performance liquid chromatography to obtain peptides with a purity of up to 95%.

[0029] 1.3 Structural Analysis

[0030] The secondary structure of the peptides was characterized by circular dichroism spectroscopy (CD). A circular polarization spectroscopy system (JASCO) was used. The peptides were dissolved in water and 50% trifluoroethanol, and CD spectra were measured in the range of 190 nm to 260 nm.

[0031] 1.4 Cytotoxicity assay

[0032] The cytotoxic effects of P-1 to P-4 on MARC-145 cells were investigated using the MTS cell viability assay. 1×10⁶ cells of MARC-145 cells in good growth condition and in the logarithmic growth phase were used. 6 Cell / mLMARC-145 cells were seeded in 96 cell culture plates and cultured for 24 h until the cells reached 80%–90% confluence. Peptides P-1, P-2, P-3, and P-4 were serially diluted from 100 μg / mL with cell maintenance medium. After discarding the supernatant, 100 μL was added to each well, and the cells were cultured in a cell incubator for 24 h. Then, 10 μL of LMTS solution was added to each well, and the cells were cultured for another 1–3 h. The absorbance at 490 nm was measured using a microplate reader, and the percentage of relative cell viability was calculated.

[0033] 1.5 Antiviral Experiment

[0034] 1.5.1 Real-time quantitative polymerase chain reaction (RT-qPCR)

[0035] MARC-145 cells were divided at a ratio of 1×10 6 Cells were seeded at a density of 1 cell / mL into 6-well plates and then treated with peptides P-1, P-2, P-3, P-4 and HP-PRRSV. After 18 hours, RNA was extracted from the cells, and a reverse transcription system was prepared to reverse transcribe the RNA into cDNA. After the reaction, the cDNA was detected by Real-time PCR. The detection system was prepared, and the primers are shown in Table 1. Each sample was tested in triplicate, and the procedure was followed. After the reaction, the data were analyzed.

[0036] Table 1 RT-qPCR primers

[0037]

[0038] 1.5.2 Western blotting (WB)

[0039] MARC-145 cells were seeded in six-well plates. When the cells reached 70% confluence, they were treated. After 18 hours, the culture medium was discarded, and the cells were treated with 5× loading solution and denatured at 100°C for 10 minutes. The denatured cells were then added to the wells of a protein gel, and the voltage was adjusted to 80V until the end of the treatment. The protein was transferred to an NC membrane by electroporation. After electroporation, the membrane was blocked with 5% skim milk for 2 hours. Primary antibody (PRRSV-N protein antibody diluted 1:1000) was prepared and incubated overnight at 4°C. The antibody was then recovered, and the membrane was washed five times with TBST for 3 minutes each time. HRP-labeled anti-rabbit IgG antibody (diluted 1:2000) was added and incubated at room temperature for 1 hour. The antibody was then recovered, and the membrane was washed five times with TBST. The chemiluminescence buffer was prepared, and the membrane was placed in a chemiluminescence imaging system for detection.

[0040] 1.5.3 Indirect immunofluorescence assay (IFA)

[0041] Take a 6-well plate and mix MARC-145 cells at a ratio of 1×10⁻⁶. 5Seed cells at a density of [number] cells / mL. When cells reach 70% confluence, treat them. After 18 hours, remove cells and discard the original culture medium. Wash cells three times with PBS for 3 minutes each time. Fix cells with 4% paraformaldehyde for 15 minutes, then wash with PBS. Permeate cells with 0.1% Triton X-100 at room temperature for 15 minutes, then wash with PBS three times. Block with 5% BSA at 37°C for 1 hour, then discard. Prepare primary antibody (1:100 dilution), 500 μL per well, incubate overnight at 4°C, then wash with PBS. Prepare secondary antibody (1:500 dilution), 500 μL per well, protect from light, incubate at 37°C for 1 hour, then discard, and wash cells five times with PBS. Add DAPI to each well and incubate at room temperature for 10 minutes, then discard. Wash cells three times with PBS and observe under a fluorescence microscope.

[0042] 2 Results Analysis

[0043] 2.1 Design and synthesis of anti-PRRSV peptides

[0044] Based on the structural features of P-1 and the membrane structure of PRRSV, P-1 was cationicized to design and synthesize three derivatives, P-2, P-3 and P-4, whose sequences are shown in Table 2.

[0045] Table 2. Peptide sequences and their physicochemical properties

[0046]

[0047] a: Molecular weight (MW) of peptides were measured by ESI-MS.

[0048] b: Retention time of peptides were determined by RP-HPLC.

[0049] 2.2 Peptide Purity and Structure Analysis

[0050] The peptides in Table 2 were synthesized using solid-phase synthesis technology, and then purified and analyzed for purity using high-performance liquid chromatography (HPLC), yielding peptides with a purity of up to 95%. Figure 1a-d). The synthesized peptides were analyzed using circular dichroism spectroscopy. The results showed that all four peptides exhibited negative peaks at approximately 200 nm in water, indicating random coiling, and positive peaks at approximately 220 nm, demonstrating certain α-helix and β-sheet structural features. Trifluoroethanol (TFE) is hydrophobic; using 50% TFE to simulate protein behavior in a cell membrane environment, significant changes were observed in the CD spectra of the four peptide samples, showing positive peaks at 200 nm and negative peaks at approximately 220 nm. This change indicates that the TFE environment induced significant changes in peptide structure, promoting the stabilization of more α-helix structures in the four peptides within the cell membrane environment. Figure 1e -f).

[0051] 2.3 Cytotoxicity

[0052] Cytotoxicity assays were performed on peptides P-1, P-2, P-3, and P-4 using the MTS method. The results showed that peptides P-1, P-2, P-3, and P-4 exhibited low toxicity to Marc-145 cells within their effective concentration range. P-1 and P-2 showed cell viability greater than 90% at a final concentration of 100 μg / mL, while P-3 and P-4 showed cell viability greater than 90% at a final concentration of 25 μg / mL. Figure 2 a). The above results were corroborated by staining with live and dead cells; staining was performed for three consecutive days, and the results were consistent. Figure 2 Therefore, 100 μg / mL was chosen for P-1 and P-2, and 25 μg / mL was chosen for P-3 and P-4 as the highest concentrations for antiviral evaluation.

[0053] 2.4 Evaluation of antiviral activity

[0054] 2.4.1 The peptide has a direct inactivating effect on HP-PRRSV.

[0055] To investigate the direct viral inactivation effects of peptides P-1, P-2, P-3, and P-4 on HP-PRRSV, HP-PRRSV was incubated with P-1 (100 μg / mL), P-2 (100 μg / mL), P-3 (25 μg / mL), and P-4 (25 μg / mL) at 37°C for 2 h. The incubated HP-PRRSV was then used to treat MARC-145 cells, and the antiviral activity of the peptides was determined by detecting viral titers.

[0056] RT-qPCR assays showed that when the virus was treated with P-1 at a concentration of 100 μg / mL, the HP-PRRSV infectivity inhibition rate was only 46.2%. When the virus was treated with P-2 at a concentration of 100 μg / mL and P-3 and P-4 at concentrations of 25 μg / mL, the highest inhibition rates against HP-PRRSV were 83.4%, 83.92%, and 80.39%, respectively. Figure 3 a). Western blotting revealed that P-2, P-3, and P-4 significantly reduced the intracellular HP-PRRSV level in MARC-145 cells. Figure 3 (b) and (c) indicate that we successfully optimized the structure of P-1, and its derived peptides P-2, P-3, and P-4 exhibited good antiviral activity, with lower concentrations demonstrating better efficacy. We then performed indirect immunofluorescence experiments (...). Figure 3 d) The results showed that peptides P-2, P-3, and P-4 had a significant direct inactivation effect on HP-PRRSV.

Claims

1. Use of a polypeptide having activity against Porcine Reproductive and Respiratory Syndrome virus (PRRSV) in the preparation of a medicament against high pathogenicity porcine reproductive and respiratory syndrome virus (HP-PRRSV), wherein the amino acid sequence of the polypeptide is as shown in SEQ ID NO. 3.

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