Foot and mouth disease virus polypeptide inhibitor and application thereof

Abstract

The invention provides a foot-and-mouth disease virus polypeptide inhibitor and application thereof. The amino acid sequence of polypeptide for inhibiting or preventing foot-and-mouth disease virus is shown as 12th-25th sites of a sequence 1. The polypeptide can specifically and effectively inhibit the replication of FMDV in cells, and can inhibit PK-15 cytopathy caused by FMDV, and the degree of inhibiting the lesion is positively correlated with the concentration of the polypeptide.

Description

A foot-and-mouth disease virus polypeptide inhibitor and its application Technical Field

[0001] The present invention relates to a foot-and-mouth disease virus polypeptide inhibitor and application thereof. Background Art

[0002] Foot-and-mouth disease virus (FMDV) belongs to the genus Aphthovirus in the family Picornaviridae. There are seven serotypes: O, A, C, Asia1, SAT1, SAT2, and SAT3. There is no cross-immunity between serotypes. FMDV is an essential tool for the early prevention and control of FMD outbreaks.

[0003] Currently, the prevention and control of foot-and-mouth disease (FMD) relies primarily on vaccines. Although vaccination provides protection against FMDV, the rapid replication and spread of the virus leaves a "window of susceptibility" after vaccination. It typically takes seven days for vaccine-derived immune protection to take effect. Targeting FMDV replication during the early stages of infection is a useful strategy to cover the window of susceptibility and prevent viral shedding; however, existing anti-FMDV treatments are limited.

[0004] The nonstructural protein 2C of FMDV is essential for viral replication. The C-terminal helical region contains a unique loop (PBL) that occupies a hydrophobic pocket in adjacent FMDV 2C molecules. Studies have shown that the PBL-pocket interaction is crucial for FMDV 2C self-oligomerization, ATPase activity, and FMDV survival. Based on this, we designed a PBL-containing peptide (PBL-peptide). This peptide can specifically inhibit FMDV replication and has broad application prospects.

[0005] Invention Disclosure

[0006] The present invention provides a foot-and-mouth disease virus polypeptide inhibitor and application thereof.

[0007] The present invention provides a polypeptide, the amino acid sequence of the polypeptide is shown in positions 12 to 25 of sequence 1 in the sequence listing.

[0008] The present invention also provides a derivative of the polypeptide, which is as follows (1) or (2):

[0009] (1) Derivatives obtained by inserting and / or replacing and / or deleting one or more amino acids in the polypeptide;

[0010] (2) a derivative obtained by adding a functional peptide to one end of the polypeptide;

[0011] (3) A derivative obtained by linking the polypeptide or the derivative described in (1) to a carrier.

[0012] The functional peptide in (2) is a membrane-penetrating peptide.

[0013] The derivative in (2) is a polypeptide having an amino acid sequence as shown in Sequence 1 of the sequence listing.

[0014] The use of the polypeptide in the preparation of foot-and-mouth disease virus inhibitors should also be within the scope of protection of the present invention.

[0015] The use of the derivatives in the preparation of foot-and-mouth disease virus inhibitors should also be within the scope of protection of the present invention.

[0016] The present invention also provides a foot-and-mouth disease virus inhibitor, the active ingredient of which is the polypeptide or the derivative.

[0017] The use of the polypeptide in the preparation of drugs for treating or preventing foot-and-mouth disease virus should also be within the scope of protection of the present invention.

[0018] The use of the derivatives in the preparation of drugs for treating or preventing foot-and-mouth disease virus should also be within the scope of protection of the present invention.

[0019] The present invention also provides a medicine for treating or preventing foot-and-mouth disease virus, the active ingredient of which is the polypeptide or the derivative.

[0020] The present invention also provides a method for treating or preventing foot-and-mouth disease virus, comprising the step of using the drug for treating or preventing foot-and-mouth disease virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG1 is a graph showing the results of a biolayer interferometry (BLI) experiment on the interaction between PBL-peptide and FMDV 2C.

[0022] Figure 2 shows an ATP inhibition experiment in the presence of PBL-peptide (1.25 μM, 12.5 μM). The amount of free phosphate was measured at different time points, and the data were linearly fitted to calculate the hydrolysis rate.

[0023] Figure 3 shows the cell permeability of TAT-PBL-peptide. PK-15 cells were incubated with 5 μM FITC-labeled TAT-PBL-peptide for 12 hours and then examined under a fluorescence microscope.

[0024] Figure 4 shows that TAT-PBL-peptide disrupts FMDV 2C-induced intracellular lipid droplet aggregation. PBL-peptide can disrupt FMDV 2C-induced LD aggregation in PK-15 cells at low micromolar concentrations (1 μM and 10 μM).

[0025] Figure 5 shows that PBL-peptide was diluted to a final concentration of 800 to 0.09 μM and added to PK-15 cells. The cells were incubated for 24 hours and the cell viability was detected using a cell counting kit-8 (CCK-8). The CC of PBL-peptide was calculated based on the results. 50 .

[0026] FIG6 shows the pathological effects of PBL-peptide added to PK-15 cells infected with FMDV (MOI=0.5) at dilution ratios with a final concentration of 400 to 3.125 μM, and observed under a microscope for 18 hours.

[0027] Figure 7 shows the dose-dependent inhibitory effect of PBL-peptide on FMDV infection in PK-15 cells, as measured by a plaque assay. Cells were seeded in 12-well plates and infected with FMDV (MOI of 0.01). TAT-PBL-peptide was diluted from 400 to 3.125 μM and added to the infected cells. Four hours after peptide addition, a plaque assay was performed.

[0028] Figure 8 shows the effect of TAT-PBL-peptide on RNA levels in PK-15 cells after FMDV infection. PK-15 cells were inoculated in 24-well plates and infected with FMDV (MOI = 0.5). The peptide was added to the cells and diluted in a concentration range of 400 to 3.125 μM. 18 hours after infection, total RNA was extracted from infected cells, cDNA was reverse transcribed, and quantified by qPCR. The GAPDH gene was used as an internal control. All data are based on at least two independent experiments. The IC value of PBL-peptide was calculated based on the results. 50 .

[0029] Figure 9 shows the effect of the control peptide TAT-peptide on FMDV viral RNA levels in PK-15 cells. PK-15 cells were inoculated in 24-well plates and infected with FMDV (MOI = 0.5). The peptide was added to the cells and diluted in series at concentrations ranging from 400 to 3.125 μM. 18 hours after infection, total RNA was extracted from infected cells, reverse transcribed into cDNA, and quantified by qPCR. The GAPDH gene was used as an internal control. The IC value of TAT-peptide was calculated based on the results. 50 .

[0030] Figure 10 shows PK-15 cells infected with FMDV (MOI = 0.5) and then treated with PBL-peptide diluted from 400 to 0 μM. After 18 hours of infection, the infected cells were treated and Western blot analysis was performed to detect differences in FMDV VP1 protein expression.

[0031] Best Mode for Carrying Out the Invention

[0032] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0033] The relevant experiments described in the following examples obtained biosafety licenses and foot-and-mouth disease experimental activity licenses: The Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences obtained a license from the Ministry of Agriculture and Rural Affairs to engage in highly pathogenic animal pathogenic microorganism experimental activities in accordance with the relevant requirements of biosafety level 3 laboratories (BSL-3) and biosafety of foot-and-mouth disease experimental activities. After applying for permission at each level, the license has been filed with the Ministry of Agriculture and Rural Affairs and meets the national biosafety level requirements.

[0034] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are commercially available unless otherwise specified.

[0035] Example 1: Binding experiment of peptide inhibitor PBL-peptide and foot-and-mouth disease virus 2C protein

[0036] 1. Preparation of PBL-peptide

[0037] The polypeptide PBL-peptide was prepared by chemical synthesis. The amino acid sequence of the PBL-peptide is shown in positions 12-25 of SEQ ID NO: 1.

[0038] 2. Construction of FMDV 2C-ΔN plasmid: The sequence between BamHI and SalI of plasmid pET-32a (Zhifan Bio) was replaced with the sequence of the FMDV 2C-ΔN gene, while keeping other sequences unchanged. The resulting recombinant vector FMDV 2C-ΔN has the sequence of the FMDV 2C-ΔN gene at positions 289-954 of GenBank ID: CAB62902.1.

[0039] 3. Preparation of FMDV 2C-ΔN protein:

[0040] The constructed recombinant expression vector pET32a was transformed into Escherichia coli BL21 (DE3), and ampicillin resistance was screened to obtain positive single clones. The obtained positive single clones were inoculated into LB liquid medium containing 100 μg / mL ampicillin, and the plasmids were extracted and sequenced. The results showed that the extracted plasmids were correct, indicating that the constructed recombinant bacteria were correct, and were recorded as recombinant bacteria BL21-pET32a-FMDV 2C-ΔN.

[0041] The recombinant strain BL21-pET32a-FMDV 2C-ΔN was inoculated into LB liquid medium containing 100 μg / mL ampicillin and shaken at 200 rpm at 37°C overnight for 12 hours to obtain a seed solution. One mL of the seed solution was inoculated into 1 L of LB liquid medium containing 100 μg / mL ampicillin and shaken at 250 rpm at 37°C until the A600 of the culture reached 0.6. IPTG was then added to a final concentration of 1 mM and the culture was shaken at 200 rpm at 18°C ​​overnight. The cells were then harvested by centrifugation at 5000 rpm for 10 minutes at 4°C. The cells were resuspended in lysis buffer (50 mM HEPES pH = 7.3, 300 mM NaCl) and disrupted by sonication at 200 W in an ice bath (3 seconds on, 5 seconds off) until the suspension became clear. The supernatant was then collected by centrifugation at 20,000 rpm for 60 minutes at 4°C. The supernatant was purified in a 5000 rpm ionizer. The supernatant was gravity-eluted through a nickel column (GE) (approximately 3 mL column volume) and washed with 30 column volumes (approximately 100 mL) of wash buffer (50 mM HEPES pH 7.3, 300 mM NaCl, 20 mM IM). Approximately 5 mL of wash buffer was retained and used to block the nickel column. TEV enzyme (Sigma) was then added overnight to remove the N-terminal fusion protein. The next day, the column was gravity-eluted, and approximately 10 column volumes of wash buffer were added to the nickel column and collected. Molecular sieve purification was performed using an ATKA pure (GE) Superdex 20010 / 300GL column (GE Healthcare) pre-equilibrated with molecular sieve buffer (20 mM HEPES pH 7.3, 100 mM NaCl, and 5 mM DTT) and eluted using a molecular sieve program. The FMDV 2C-ΔN protein was obtained.

[0042] Preparation of LB liquid culture medium containing 100 μg / mL ampicillin: Add ampicillin to the LB liquid culture medium so that the concentration of ampicillin in the LB liquid culture medium is 100 μg / mL. The resulting mixed solution is an LB liquid culture medium containing 100 μg / mL ampicillin.

[0043] LB liquid culture medium composition: composed of yeast extract, peptone, NaCl and water, with the concentration of each component in the solution being: yeast extract 0.5% (mass percentage), peptone 1% (mass percentage), NaCl 1% (mass percentage).

[0044] 4. Determination of the binding affinity between the peptide inhibitor PBL-peptide and the foot-and-mouth disease virus 2C protein

[0045] The direct binding affinity of PBL-peptide to the foot-and-mouth disease virus 2C protein (FMDV 2C) was assessed using biolayer interferometry (BLI). Using an Octet RED96 instrument (ForteBio), N-terminally biotinylated PBL-peptide (Biotin-PBL-peptide) was synthesized and immobilized on a streptavidin (SA) biosensor. The biotinylated peptide was then bound to 1.25–10 μM FMDV 2C-ΔN protein in solution. All experiments were performed at 25°C and 1000 rpm. 200 nM of the biotinylated PBL-peptide was loaded onto the streptavidin (SA) biosensor probe pre-equilibrated in PBS buffer. In a buffer solution containing 25 mM MES buffer (pH 5.5), 100 mM NaCl, and 0.02% surfactant P20 (Cytiva), the peptide was bound to 1.25 μM, 2.5 μM, 5 μM, and 10 μM FMDV 2C-ΔN, respectively. Association and dissociation plots were recorded (as shown in Figure 1 , where 1.25 μM, 2.5 μM, 5 μM, and 10 μM represent the binding and dissociation of the peptide at 1.25 μM, 2.5 μM, 5 μM, and 10 μM peptide concentrations, respectively; 1.25 μM Fit, 2.5 μM Fit, 5 μM Fit, and 10 μM Fit represent the binding and dissociation of the peptide at 1.25 μM, 2.5 μM, 5 μM, and 10 μM peptide concentrations, respectively, after a 1:1 global fit). Double reference subtraction was used to remove the effects of baseline drift and nonspecific binding. The association rate constant, Ka, and dissociation rate constant, Kd, ​​of the binding reaction were given. Data Analysis 11.1 software was used to perform a global fit of multiple kinetic trajectories to a 1:1 binding model, yielding the equilibrium dissociation constant, KD. The affinity of PBL-peptide for FMDV 2C-ΔN was in the nanometer range, with an equilibrium dissociation constant, KD, of 276 nM, indicating a high potency of the PBL-peptide. Notably, the binding kinetics of PBL-peptide to FMDV 2C exhibited a rapid binding rate (Ka = 2290 M -1s-1 ) and a slow dissociation rate (Kd = 6.32*10 -4 s -1 ), a property that is important not only for the binding affinity to the target but also for the extension of the inhibition half-life.

[0046] Example 2: PBL-peptide inhibits FMDV 2C ATPase activity in vitro

[0047] 1. Construction of MBP-FMDV 2C plasmid:

[0048] The sequence between BamHI and HindIII of the vector pMal-C2X (Aono gene) was replaced with the coding gene sequence of FMDV 2C, and the coding gene sequence of FMDV 2C was the sequence shown in GenBank ID: CAB62902.1.

[0049] 2. Preparation of MBP FMDV 2C protein:

[0050] The constructed recombinant expression vector pMal-C2X was transformed into Escherichia coli BL21 (DE3), and ampicillin resistance was screened to obtain positive single clones. The obtained positive single clones were inoculated into LB liquid medium containing 100 μg / mL ampicillin, and the plasmids were extracted and sequenced. The results showed that the extracted plasmids were correct, indicating that the constructed recombinant bacteria were correct, and were recorded as recombinant bacteria BL21-MBP-FMDV 2C.

[0051] The recombinant strain BL21-MBP-FMDV 2C was inoculated into LB liquid medium containing 100 μg / mL ampicillin and shaken at 200 rpm at 37°C overnight for 12 hours to obtain a seed solution. 1 mL of the seed solution was inoculated into 1 L of LB liquid medium containing 100 μg / mL ampicillin and shaken at 250 rpm at 37°C until the A600 of the culture reached 0.6. IPTG was then added to a final concentration of 1 mM and the culture was shaken at 200 rpm at 18°C ​​overnight. The cells were then collected by centrifugation at 5000 rpm for 10 minutes at 4°C. The cells were resuspended in lysis buffer (50 mM HEPES pH=7.3, 300 mM NaCl) and disrupted by sonication at 200 W on ice (3 seconds on, 5 seconds off) until the suspension became clear. The supernatant was then collected by centrifugation at 20,000 rpm at 4°C for 60 minutes. The supernatant was purified in a 5000 rpm ionizer. The supernatant was eluted by gravity through a nickel column (GE) (column volume approximately 3 mL), washed with 30 column volumes (approximately 100 mL) of wash buffer (50 mM EPES pH = 7.3, 300 mM NaCl, 20 mM IM), and eluted with approximately 10 column volumes of eluent (50 mM HEPES pH = 7.3, 300 mM NaCl, 20 mM maltose), and the eluate was collected. Molecular sieve purification was performed using an ATKA pure (GE) Superdex 200 10 / 300GL column (GE Healthcare) pre-equilibrated with molecular sieve buffer (20 mM HEPES pH 7.3, 100 mM NaCl, and 5 mM DTT) and eluted using a molecular sieve program. MBP FMDV 2C protein was obtained.

[0052] 3. Thin layer chromatography (TLC) was used to separate the free γ- 32 P is separated from ADP, and the hydrolysis ratio of FMDV 2C ATPase is measured using autoradiography. The amount of substrate hydrolyzed is calculated based on the total amount of substrate added and the measured hydrolysis ratio. Since the reaction time is known, the Michaelis-Menten equation for the native protein can be calculated, as well as the substrate hydrolysis concentration per unit time under certain substrate and enzyme concentrations. This allows the inhibitory effect of the PBL-peptide on FMDV 2C ATPase activity to be determined. The specific steps are as follows:

[0053] 1) Determine the experimental system: the reaction system is 50 μL, including 10 μL of 5X reaction buffer (20 mM Mg(CH3COO)2, 100 mM HEPES / KOH pH 7.0, 25 mM DTT), γ- 32 The P stock solution was 1 μL, the final concentration of ATPase (MBP-FMDV 2C) was 1.25 μM, the final concentration of ATP substrate was 500 μM, PBL-peptide (1.25 μM and 12.5 μM), and no PBL-peptide (0 μM) was set as a control. The specific components of the three reaction systems are shown in Table 1 below.

[0054] Table 1. Specific components of the three reaction systems

[0055] Ingredients 1235 X Reaction buffer10μL10μL10μLγ- 32 P-ATP 1 μL 1 μL 1 μL ATPase (FMDV 2C) Final concentration is 1.25 μM Final concentration is 1.25 μM Final concentration is 1.25 μM

[0056] The final concentration of PBL-peptide is 1.25 μM. The final concentration is 12.5 μM. The final concentration is 0 μM. The final concentration of ATP is 500 μM. The final concentration is 500 μM. The total volume is 50 μL. The total volume is 50 μL.

[0057] (2) Chromatography plate selection: Polyethyleneimine (PEI)-cellulose plate (Sigma) was used. Each plate was cut to a width of 10 cm. A dotted line was drawn with a pencil approximately 1.5 cm from the bottom, with each sample spaced 1 cm apart.

[0058] (3) Prepare the developing agent: The chromatography fluid consists of 0.8 M acetic acid and 0.8 M lithium chloride.

[0059] (4) Reaction: Prepare the reaction solution according to the reaction system and react at 30°C. Samples were taken at 2 min, 4 min, 6 min, and 8 min, and EDTA was added to a final concentration of 0.6 M to terminate the reaction.

[0060] (5) Spotting: Pipette 1.2 μL of the reaction solution and spot it on the chromatography plate, with each sample spaced 1 cm apart.

[0061] (6) Development: Place the chromatography plate in a chromatography tank containing 25 mL of chromatography fluid for chromatography. When the chromatography fluid develops to 1.5 cm from the top, take out the chromatography plate and place it on clean filter paper to dry naturally or blow dry with a hair dryer.

[0062] (7) Screen pressing: Wrap the dried chromatography plate with plastic wrap, and then press the phosphor screen onto the chromatography plate for at least 2 hours.

[0063] (8) Data processing: Use Typhoon scanner to scan the phosphor screen to obtain the image, and then use ImageQuantTL to quantify the gray value and calculate the hydrolysis amount.

[0064] The results are shown in Figure 2. In Figure 2, 0 μM PBL-peptide represents ATP hydrolysis by MBP-FMDV 2C; 1.25 μM PBL-peptide represents ATP hydrolysis by MBP-FMDV 2C in the presence of 1.25 μM PBL-peptide; and 12.5 μM PBL-peptide represents ATP hydrolysis in the presence of 12.5 μM PBL-peptide. Figure 2 shows that PBL-peptide inhibits the ATP hydrolysis ability of MBP-FMDV 2C in a concentration-dependent manner.

[0065] Example 3: Detection of TAT-PBL-peptide membrane penetration efficiency

[0066] 1. Preparation of TAT-PBL-peptide

[0067] The cell-penetrating peptide TAT was added to the N-terminus of PBL-peptide, and the new peptide was named TAT-PBL-peptide (N'-YGRKKRRQRRRNLHEKVASQPIFKQ-C', as shown in Sequence 1 in the sequence listing). To observe the cell-penetrating effect of the peptide, FITC was added to the N-terminus of TAT-PBL-peptide and named: FITC-TAT-PBL-peptide.

[0068] 2. TAT-PBL-peptide membrane penetration efficiency detection

[0069] Experimental method: Fluorescence microscopy observation

[0070] In this example, porcine kidney cells (PK-15, purchased from the Cell Resource Center, IBMS, CAMS / PUMC) were used as the research subjects. MEM complete medium (purchased from Gibco, supplemented with 10% fetal bovine serum and 100 mg / mL streptomycin) was prepared. PK-15 cells were plated in 12-well plates in advance. After the cells grew into a monolayer, MEM complete medium containing 5 μM FITC-TAT-PBL-peptide was added. After 12 hours, the cells were washed three times with PBS. The peptide incorporation into the cells was observed under a fluorescence microscope and a confocal microscope. The results are shown in Figure 3. In Figure 3, the left panel (Mock) shows cell fluorescence without peptide addition. Brightfield indicates brightfield fluorescence, and FITC indicates darkfield fluorescence. FITC-TAT-PBL-peptide shows cell fluorescence in the presence of 5 μM FITC-TAT-PBL-peptide. Brightfield indicates brightfield fluorescence, FITC indicates darkfield fluorescence, and superomise indicates the superposition of the two. The image on the right shows the peptide entering cells as observed under a confocal microscope. This experiment demonstrates that the TAT-PBL-peptide has good cell permeability and can penetrate the cell membrane.

[0071] Example 4: PBL-peptide disrupts FMDV 2C-induced lipid droplet aggregation in cells.

[0072] 1. Construction of N-mCherry-FMDV 2C Plasmid

[0073] The sequence between NdeI and BamHI of plasmid pCDNA 3.1 (Thermo Fisher Scientific) was replaced with the mCherry coding gene and the FMDV 2C coding gene. The FMDV 2C coding gene sequence is shown in GenBank ID: CAB62902.1, and the mCherry coding gene is shown in GenBank ID: FM169983.1.

[0074] 2. Experiments on the disruption of FMDV 2C-induced lipid droplet aggregation in cells by PBL-peptide

[0075] In order to identify whether peptide inhibitors can inhibit 2C localization and promote lipid droplet aggregation, confocal microscopy was used to observe the effect of peptides on 2C lipid droplet aggregation. The specific experimental steps are as follows:

[0076] (1) Plating: PK-15 cells were plated in a 12-well plate with a cell slide in advance, and the cells were kept aside after they grew into a monolayer the next day.

[0077] (2) Transfection: Lipofectamine™ 2000 (Invitrogen) was used as the transfection reagent, with 3 μL of transfection reagent and 2 μg of N-mCherry-FMDV 2C plasmid per well. Following the instructions for the transfection reagent, the plasmid and transfection reagent were mixed, allowed to stand for 20 min, and then added to a 12-well plate (MEM medium without antibiotics or serum had been replaced in advance). Six hours later, the medium was replaced with MEM complete medium containing 0 μM, 1 μM, and 10 μM TAT-PBL-peptide, respectively.

[0078] (3) Staining: 24 hours after transfection, cells were stained.

[0079] (4) Slide treatment: Add 1.2 μL of anti-fluorescence quencher to the microscope slide in advance, and then turn the cell slide upside down onto the slide.

[0080] (5) Confocal microscopy: Observe the slides using a confocal microscope.

[0081] The results are shown in Figure 4 (left figure: 2C localization and lipid droplet aggregation in the presence of 0 μM, 1 μM and 10 μM TAT-PBL-peptide, respectively, mCherry indicates the expression of 2C, Bodipy493 / 503 indicates the expression of intracellular lipid droplets, merge indicates the co-localization of the two, and zoom indicates the magnification of the white box in the merge figure. The right figure shows the average number of intracellular lipid droplets in the presence of different concentrations of PBL-peptide.) It can be seen from Figure 4 that TAT-PBL-peptide can disrupt the intracellular lipid droplet aggregation induced by FMDV 2C, and TAT-PBL-peptide can disrupt the aggregation of FMDV 2C-induced LDs in PK-15 cells at low micromolar concentrations (1 μM and 10 μM).

[0082] Example 5: PBL-peptide cytotoxicity assay.

[0083] In the antiviral process, the peptide PBL-peptide must not only inhibit the virus but also ensure that it is non-toxic to cells. Therefore, this indicator is tested through cytotoxicity testing, with untreated cells as the control group.

[0084] PK-15 cells were plated in a 96-well plate, with 100 μL added to each well. When cells reached 70%-80% confluency, the MEM medium supplemented with 10% serum was replaced with a gradient of PBL-peptide concentrations, resulting in final PBL-peptide concentrations of 0.097 μM, 0.19 μM, 0.39 μM, 0.78 μM, 1.56 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, 200 μM, 400 μM, and 800 μM, respectively. 24 hours after peptide addition, 10 μL of the live cell assay reagent CCK-8 was added to each well and mixed thoroughly. The cells were incubated at 37°C for 4 hours. The absorbance at OD450 nm was measured using a microplate reader. The results are shown in Figure 5. The viable cell count of untreated cells is 100%. After adding 100 μM peptide, the viable cell count is basically the same as that of the control group (untreated cells), which proves that the CC of the peptide PBL-peptide is 50 >800μM.

[0085] Example 6: Determination of the antiviral efficiency of PBL-peptide.

[0086] The FMDV O / BY / CHA / 2010 strain used in the example is preserved by the National Foot-and-Mouth Disease Reference Laboratory designated by the Veterinary Bureau of the Ministry of Agriculture and Rural Affairs. The public can obtain it through a letter of authorization approved by the Veterinary Bureau of the Ministry of Agriculture and Rural Affairs. It is referred to as FMDV in this example.

[0087] 1. Cytopathic Effect Experiment

[0088] PK-15 cells were seeded in a 96-well plate. After the cells formed a monolayer, 100 μL of FMDV (MOI = 0.5) was added to each well. After incubation at 37°C for 2 hours, the virus solution was discarded. PBL-peptide was diluted in complete MEM medium and added to the infected cells, resulting in final PBL-peptide concentrations of 0 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, 200 μM, and 400 μM in the wells. A normal cell control was also established. Cytopathic effects were observed microscopically 18 hours later. As shown in Figure 6, the PBL-peptide peptide inhibited FMDV-induced PK-15 cytopathic effects, and the degree of inhibition was positively correlated with peptide concentration.

[0089] 2. Plaque Formation Experiment

[0090] PK-15 cells were seeded in 12-well plates and cultured for 48 hours. FMDV (MOI = 0.01) was then inoculated and incubated at 37°C for 1 hour, with the plates shaken every 10 minutes. The virus solution was then discarded, and PBL-peptide diluted in complete MEM medium was added to achieve final PBL-peptide concentrations of 0 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, 200 μM, and 400 μM in each well. Untreated normal cells were also included as controls. The cells were incubated at 37°C for 4 hours, and the supernatant was discarded. Plaque assays were performed. As shown in Figure 7, the PBL-peptide peptide reduced the number of plaques formed in PK-15 cells induced by FMDV replication, and the degree of plaque reduction was positively correlated with peptide concentration.

[0091] 3. RT-qPCR determination of viral RNA and calculation of 50% inhibitory concentration (IC 50 )

[0092] PK-15 cells were seeded in 24-well plates. After the cells grew to a monolayer, they were infected with FMDV (MOI = 0.5). After incubation at 37°C for 2 hours, peptides diluted in MEM medium were added so that the final PBL-peptide concentrations in the wells were 0 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, 200 μM, and 400 μM, respectively. A normal cell control was also set up. 18 hours after infection, the supernatant was discarded, and total RNA was extracted from the infected cells. After reverse transcription of cDNA, the difference in FMDV replication levels was determined by fluorescence quantitative PCR, with the GAPDH gene as an internal reference. IC values ​​were calculated based on the qPCR results using Graphpad Prism9 software. 50 The results show that the peptide PBL-peptide can effectively inhibit the replication of FMDV, and its IC 50 It is 5.526μM.

[0093] In order to verify the specificity of PBL-peptides in inhibiting FMDV viral replication in cells and that it was not caused by the membrane-penetrating peptide TAT, the same method was used to detect the accumulation of viral RNA in infected cells after treatment with the control peptide TAT-peptides (amino acid sequence 1-11 of sequence 1), and the IC of the control peptide was calculated. 50 The concentration of TAT-PBL-peptide was about 72 μM (as shown in Figure 9), which was much higher than that of TAT-PBL-peptide. Therefore, the effect of TAT on viral inhibition was excluded.

[0094] 4. Western Blot Determination of Viral Protein Expression

[0095] PK-15 cells were seeded in 6-well plates and infected with FMDV (MOI = 0.5). Two hours later, peptides diluted in MEM medium were added, resulting in final PBL-peptide concentrations in the wells of 0 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, 200 μM, and 400 μM, respectively. A normal cell control was also established. Samples were collected 18 hours after infection and analyzed by Western blot for viral VP1 protein expression. The results showed a significant decrease in FMDV protein levels with increasing peptide concentration (Figure 10), demonstrating that the PBL-peptide peptide can inhibit FMDV replication.

[0096] In summary, our study provides a peptide PBL-peptide and proves that the peptide can specifically and effectively inhibit the replication of FMDV in cells.

[0097] Although the above describes the specific embodiments of the present invention in conjunction with the accompanying drawings, it is not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art on the basis of the technical solution of the present invention without any creative work are still within the scope of protection of the present invention.

[0098] Industrial Applications

[0099] The present invention provides a polypeptide PBL-peptide, which can specifically and effectively inhibit the replication of FMDV in cells and can inhibit the pathological changes of PK-15 cells caused by FMDV, and the degree of inhibition of pathological changes is positively correlated with the concentration of the polypeptide.

Claims

1. A polypeptide, the amino acid sequence of which is shown in positions 12-25 of SEQ ID NO: 1 in the sequence listing.

2. The derivative of the polypeptide according to claim 1, which is as follows (1) or (2) or (3): (1) Derivatives obtained by inserting and / or replacing and / or deleting one or more amino acids of the polypeptide according to claim 1; (2) A derivative obtained by adding a functional peptide to one end of the polypeptide according to claim 1; (3) A derivative obtained by linking the polypeptide according to claim 1 or the derivative according to (1) to a carrier.

3. The derivative according to claim 2, characterized in that The functional peptide in (2) is a membrane-penetrating peptide.

4. The derivative according to claim 3, characterized in that The derivative in (2) is a polypeptide having an amino acid sequence as shown in Sequence 1 of the sequence listing.

5. Use of the polypeptide according to claim 1 in the preparation of a foot-and-mouth disease virus inhibitor or a drug for preventing foot-and-mouth disease virus.

6. A foot-and-mouth disease virus inhibitor, the active ingredient of which is the polypeptide according to claim 1 or the derivative according to any one of claims 2 to 4.

7. Use of the derivative according to any one of claims 2 to 4 in the preparation of a medicament for treating or preventing foot-and-mouth disease virus or in the preparation of a foot-and-mouth disease virus inhibitor.

8. A drug for treating or preventing foot-and-mouth disease virus, wherein the active ingredient is the polypeptide according to claim 1 or the derivative according to any one of claims 2 to 4.

9. A method for treating or preventing foot-and-mouth disease virus, characterized in that: The method comprises the step of using the drug for treating or preventing foot-and-mouth disease virus according to claim 8.

Images