WO2023240458A1

WO2023240458A1 - Polypeptide inhibitor of foot-and-mouth disease virus and use thereof

Info

Publication number: WO2023240458A1
Application number: PCT/CN2022/098719
Authority: WO
Inventors: 崔胜�; 郑海学; 张楚; 杨帆; 高小攀; 张伟; 秦博; 曹伟军; 郑敏
Original assignee: 中国医学科学院病原生物学研究所; 中国农业科学院兰州兽医研究所
Priority date: 2022-06-14
Filing date: 2022-06-14
Publication date: 2023-12-21
Also published as: ZA202306089B

Abstract

Provided in the present invention are a polypeptide inhibitor of foot-and-mouth disease virus and the use thereof. The amino acid sequence of the polypeptide for inhibiting or preventing foot-and-mouth disease virus is as shown in positions 12-25 of 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 cytopathy is positively correlated with the concentration of the polypeptide.

Description

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

Technical field

The invention relates to a foot-and-mouth disease virus polypeptide inhibitor and its application.

Background technique

Foot-and-mouth disease virus (FMDV) belongs to the genus Aphthovirus in the family picornaviridae. There are serotypes O, A, C, Asia1, SAT1, SAT2 and SAT37. There is no difference between serotypes. Cross-immunity phenomenon. It is an important means for early prevention and control of the spread of foot-and-mouth disease.

At present, the prevention and treatment of foot-and-mouth disease mainly relies on vaccines. Although vaccination provides protection against FMDV, the rapid replication and spread of the virus leaves a "window of susceptibility" after vaccination. It usually takes 7 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 susceptibility window and prevent viral shedding; however, existing anti-FMDV treatments are limited.

The nonstructural protein 2C of FMDV is critical for viral replication. The C-terminal helical region contains a unique loop (PBL) that occupies a hydrophobic pocket in the adjacent FMDV 2C molecule. Studies have shown that the PBL-Pocket interaction is critical for FMDV 2C auto-oligomerization, ATPase activity, and FMDV survival. On this basis, we designed a polypeptide containing PBL (PBL-peptide). This polypeptide can specifically inhibit the replication of FMDV and has broad application prospects.

invention disclosure

The invention provides a foot-and-mouth disease virus polypeptide inhibitor and its application.

The present invention provides a polypeptide, the amino acid sequence of which is shown in positions 12-25 of Sequence 1 of the sequence listing.

The invention also provides derivatives of the polypeptide, which are as follows (1) or (2):

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

(2) Derivatives obtained by adding a functional peptide to one end of the polypeptide;

(3) Derivatives obtained by coupling the polypeptide or the derivative described in (1) to a carrier.

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

The derivative in (2) is a polypeptide whose amino acid sequence is shown in Sequence 1 of the sequence listing.

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

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

The present invention also provides a foot-and-mouth disease virus inhibitor whose active ingredient is the polypeptide or the derivative.

The application of the polypeptide in the preparation of medicines for treating or preventing foot-and-mouth disease virus should also be within the protection scope of the present invention.

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

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.

The present invention also provides a method for treating or preventing foot-and-mouth disease virus, which includes the step of using the medicine for treating or preventing foot-and-mouth disease virus.

Description of the drawings

Figure 1 shows the results of the biolayer interference analysis (BLI) experiment of the interaction between PBL-peptide and FMDV 2C.

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

Figure 3 shows the cell penetration ability of TAT-PBL-peptide. PK-15 cells were incubated with 5 μM FITC-labeled TAT-PBL-peptide for 12 hours and then examined by fluorescence microscopy.

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

Figure 5 shows that PBL-peptide was added to PK-15 cells at a final concentration of 800 to 0.09 μM and incubated for 24 hours. The cell viability was detected using Cell Counting Kit-8 (CCK-8). PBL-peptide was calculated based on the results. CC ₅₀ .

Figure 6 shows that PBL-peptide was added to PK-15 cells infected with FMDV (MOI=0.5) at a final concentration of 400-3.125 μM at multiple dilutions, and the pathological effects of PK-15 cells were observed under a microscope for 18 hours.

Figure 7 shows the dose-dependent inhibitory effect of PBL-peptide on FMDV infection detected by plaque assay in PK-15 cells. Cells were seeded in 12-well plates and infected with FMDV (MOI: 0.01). TAT-PBL-peptide was diluted from 400 to 3.125 μM and added to the infected cells. Plaque experiments were performed 4 hours after adding the peptide.

Figure 8 shows the effect of TAT-PBL-peptide on RNA levels after FMDV infection of PK-15 cells. PK-15 cells were inoculated into a 24-well plate and infected with FMDV (MOI=0.5). The polypeptide was added to the cells and diluted at a concentration of 400 to 3.125 μM. At 18 h postinfection, total RNA was extracted from infected cells, cDNA was reverse transcribed and quantified by qPCR. GAPDH gene served as internal control. All data are based on at least two independent experiments. Calculate the IC ₅₀ of PBL-peptide based on the results.

Figure 9 shows the effect of the control polypeptide TAT-peptide on FMDV viral RNA levels in PK-15 cells. PK-15 cells were inoculated into a 24-well plate and infected with FMDV (MOI=0.5). The polypeptide was added to the cells and diluted at a concentration of 400 to 3.125 μM. At 18 h postinfection, total RNA was extracted from infected cells, cDNA was reverse transcribed and quantified by qPCR. GAPDH gene served as internal control. Calculate the IC ₅₀ of TAT-peptide based on the results.

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

Best way to implement your invention

The present invention will be described in further detail below in conjunction with specific embodiments. The examples given 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 those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

The relevant experiments described in the following examples obtained biosafety licenses and foot-and-mouth disease experimental activity licenses: Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, in accordance with the relevant requirements of biosafety level three laboratories (BSL-3) and foot-and-mouth disease experimental activity biosafety, after approval We submitted an application to the Ministry of Agriculture and Rural Affairs and obtained a license from the Ministry of Agriculture and Rural Affairs to engage in experimental activities involving highly pathogenic animal pathogenic microorganisms. It has been registered with the Ministry of Agriculture and Rural Affairs and complies with the requirements of the national biosafety level.

The experimental methods in the following examples are all conventional methods unless otherwise specified. Materials, reagents, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified.

Example 1: Binding experiment between polypeptide inhibitor PBL-peptide and foot-and-mouth disease virus 2C protein

1. Preparation of polypeptide PBL-peptide

The polypeptide PBL-peptide is prepared by chemical synthesis. The amino acid sequence of the PBL-peptide is shown in positions 12-25 of Sequence 1.

2. Construction of FMDV 2C-ΔN plasmid: Replace the sequence between BamHI and SalI of plasmid pET-32a (Zhifan Biotech) with the sequence of FMDV 2C-ΔN gene, and keep other sequences unchanged to obtain the recombinant vector FMDV 2C-ΔN, the sequence of FMDV 2C-ΔN gene is GenBank ID: CAB62902.1 at positions 289-954.

3. Preparation of FMDV 2C-ΔN protein:

The constructed recombinant expression vector pET32a was transformed into Escherichia coli BL21 (DE3), screened for ampicillin resistance, and a positive single clone was obtained; the obtained positive single clone was inoculated into LB liquid medium containing 100ug/mL ampicillin and cultured, and the plasmid was extracted. , sequencing results showed that the extracted plasmid was correct, indicating that the constructed recombinant bacterium was correct, which was recorded as the recombinant bacterium BL21-pET32a-FMDV 2C-ΔN.

Inoculate the recombinant strain BL21-pET32a-FMDV 2C-ΔN into LB liquid culture medium containing 100ug/mL ampicillin, shake at 200rpm at 37℃, and culture overnight (12h) to obtain a seed liquid; take 1mL of seed liquid and inoculate it into 1L containing LB liquid culture medium with ampicillin 100ug/mL, shake at 250rpm at 37℃, until the A600 of the culture system is 0.6, add IPTG to a final concentration of 1mM, shake at 200pm at 18℃ overnight, centrifuge at 5000rpm for 10min at 4℃, collect the cells . Use lysis buffer (Lysis buffer: 50mM HEPES pH = 7.3, 300mM NaCl) to resuspend the bacterial cells, 200W power ice bath ultrasonic (ultrasound for 3 seconds, pause for 5 seconds) to break the bacterial cells until the suspension becomes clear, and then centrifuge at 4°C and 20000rpm for 60 minutes. Collect the supernatant. The supernatant was purified in a °C purification cabinet. Elute the supernatant through a nickel column (GE) by gravity (column volume is about 3mL), and use 30 times the column volume (about 100mL) of washing buffer (Wash buffer: 50mM HEPES pH=7.3, 300mM NaCl, 20mM IM) to flush the impurities; retain about 5mL of the flushing solution, plug the nickel column and add TEV enzyme (sigma) overnight to remove the N-terminal fusion protein. The next day, allow the liquid on the column to gravity elute and then add about 10 times the column Volume of impurity was passed through the nickel column and collected; molecular sieve purification was performed using ATKA pure (GE), and a Superdex 20010/300GL column (GE Healthcare) was pre-equilibrated with molecular sieve buffer (20mM HEPES pH 7.3, 100mM NaCl and 5mM DTT), using Molecular sieve program for elution. FMDV 2C-ΔN protein was obtained.

Preparation of LB liquid culture medium containing 100ug/mL ampicillin: Add ampicillin to the LB liquid culture medium so that the concentration of ampicillin in the LB liquid culture medium is 100ug/mL. The resulting mixed solution contains 100ug ampicillin. /mL of LB liquid culture medium.

LB liquid culture medium consists of yeast extract, peptone, NaCl and water. The concentration of each component in the solution is: yeast extract 0.5% (mass percentage), peptone 1% (mass percentage), NaCl 1 % (mass percentage).

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

The direct binding affinity of PBL-peptide to foot-and-mouth disease virus 2C protein (referred to as FMDV 2C) was evaluated using biolayer interference (BLI). Among them, the N-terminal biotinylated PBL-peptide (Biotin-PBL-peptide for short) was synthesized using the Octet RED96 instrument (ForteBio) and fixed on the streptavidin (SA) biosensor in the solution. Binds to 1.25-10μM FMDV 2C-ΔN protein. All experiments were performed at 25°C and 1000 rpm. Biotinylated 200 nM PBL-peptide was loaded onto a pre-equilibrated streptavidin (SA) biosensor probe in PBS buffer. In a buffer containing 25mM MES buffer (pH=5.5), 100mM NaCl and 0.02% surfactant P20 (Cytiva) (solvent is water), FMDV 2C-ΔN was mixed with 1.25μM, 2.5μM, 5μM and 10μM respectively. Bind, and record the association and dissociation diagrams (as shown in Figure 1. In Figure 1, 1.25 μM, 2.5 μM, 5 μM, and 10 μM respectively represent the binding and dissociation of the polypeptide and the protein at the polypeptide concentrations of 1.25 μM, 2.5 μM, 5 μM, and 10 μM. 1.25μM Fit, 2.5μM Fit, 5μM Fit and 10μM Fit respectively represent the binding and dissociation of the peptide and protein after global fitting at 1.25μM, 2.5μM, 5μM and 10μM peptide concentrations according to 1:1). Double reference subtraction was used to remove the effects of baseline drift and non-specific binding. The association rate constant Ka and the dissociation rate constant Kd for the binding reaction are given. Data Analysis 11.1 software was used to globally fit multiple kinetic trajectories into a 1:1 binding model, and the equilibrium dissociation constant KD was obtained. The affinity of PBL-peptide to FMDV 2C-ΔN is in the nanoscale range, and the equilibrium dissociation constant KD=276nM, indicating that the potency of PBL-peptide is relatively high. It is worth noting that the binding kinetics of PBL-peptide to FMDV 2C shows a fast binding rate (Ka=2290M ^-1s-1 ) and a slow dissociation rate (Kd=6.32*10 ^-4 s ^-1 ), which One property is important not only for binding affinity to the target, but also for prolongation of the inhibitory half-life.

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

1.Construction of MBP-FMDV 2C plasmid:

Replace the sequence between BamHI and HindIII of the vector pMal-C2X (Ono gene) with the coding gene sequence of FMDV 2C. The coding gene sequence of FMDV 2C is the sequence shown in GenBank ID: CAB62902.1.

2. Preparation of MBP FMDV 2C protein:

The constructed recombinant expression vector pMal-C2X was transformed into E. coli BL21 (DE3), and ampicillin resistance was screened to obtain a positive single clone; the obtained positive single clone was inoculated into LB liquid medium containing 100ug/mL ampicillin and cultured. The plasmid was extracted and sequenced. The results showed that the extracted plasmid was correct, indicating that the constructed recombinant bacterium was correct, which was recorded as the recombinant bacterium BL21-MBP-FMDV 2C.

Inoculate the recombinant strain BL21-MBP-FMDV 2C into LB liquid culture medium containing 100ug/mL ampicillin, shake at 200rpm at 37℃, and culture overnight (12h) to obtain a seed liquid; take 1mL of seed liquid and inoculate it into 1L containing ampicillin 100ug/mL LB liquid culture medium, shake at 250rpm at 37℃ until the A600 of the culture system is 0.6, add IPTG to a final concentration of 1mM, shake at 200pm at 18℃ overnight, centrifuge at 5000rpm for 10min at 4℃ to collect the cells. Use lysis buffer (Lysis buffer: 50mM HEPES pH = 7.3, 300mM NaCl) to resuspend the bacterial cells, 200W power ice bath ultrasound (ultrasound for 3 seconds, pause for 5 seconds) to break the bacterial cells until the suspension becomes clear, and then centrifuge at 4°C and 20000rpm for 60 minutes. Collect the supernatant. The supernatant was purified in a °C purification cabinet. Elute the supernatant through a nickel column (GE) by gravity (column volume is about 3mL), and use 30 times the column volume (about 100mL) of washing buffer (Wash buffer: 50mM EPES pH=7.3, 300mM NaCl, 20mM IM) to flush out the impurities; use about 10 times the column volume to elute (Elution buffer: 50mM HEPES pH = 7.3, 300mM NaCl, 20mM maltose) and collect the eluent. ATKA pure (GE) was used for molecular sieve purification, Superdex 200 10/300GL column (GE Healthcare) was pre-equilibrated with molecular sieve buffer (20mM HEPES pH 7.3, 100mM NaCl and 5mM DTT), and the molecular sieve program was used for elution. Obtain MBP FMDV 2C protein.

3. Use thin layer chromatography (TLC) to separate the free γ- ³² P and ADP after hydrolysis, and use autoradiography technology to determine the hydrolysis ratio of FMDV 2C ATPase. The amount of hydrolyzed substrate 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 of the natural protein can be calculated, as well as the unit time under a certain substrate concentration and enzyme concentration. The hydrolysis concentration of the substrate was measured to measure the inhibitory effect of PBL-peptide on FMDV 2C ATPase activity. Specific steps are as follows:

1) Determine the experimental system: The reaction system is 50 μL, including 10 μL of 5X Reaction buffer (20mM Mg(CH ₃ COO) ₂ , 100mM HEPES/KOH PH7.0, 25mM DTT), 1 μL of γ- ³² P stock solution, and ATPase (MBP-FMDV 2C) The final concentration is 1.25 μM, the final concentration of ATP substrate is 500 μM, PBL-peptide (1.25 μM and 12.5 μM), and no PBL-peptide (0 μM) is added as a control. The specific components of the three groups of reaction systems are as follows in Table 1 shown.

Table 1. Specific components of the three groups of reaction systems

成分 Element	11	22	33
5 X Reaction buffer5X Reaction buffer	10μL10μL	10μL10μL	10μL10μL
γ- ³²P-ATP γ- ³² P-ATP	1μL1μL	1μL1μL	1μL1μL
ATPase(FMDV 2C)ATPase(FMDV 2C)	终浓度为1.25μMThe final concentration is 1.25 μM	终浓度为1.25μMThe final concentration is 1.25 μM	终浓度为1.25μMThe final concentration is 1.25 μM

PBL-peptidePBL-peptide	终浓度为1.25μMThe final concentration is 1.25 μM	终浓度为12.5μMThe final concentration is 12.5 μM	终浓度为0μMThe final concentration is 0 μM
ATPATP	终浓度为500μM.The final concentration is 500 μM.	终浓度为500μM.The final concentration is 500 μM.	终浓度为500μM.The final concentration is 500 μM.
	总体积50μLTotal volume 50μL	总体积50μLTotal volume 50μL	总体积50μLTotal volume 50μL

(2) Select the chromatography plate: the chromatography plate is polyethyleneimine (PEI)-cellulose plate (Sigma). Cut each chromatography plate to a width of 10cm, draw dotted lines with a pencil about 1.5cm from the bottom, and space each sample 1cm wide.

(3) Prepare developing agent: The components of the chromatography solution are 0.8M acetic acid and 0.8M lithium chloride.

(4) Reaction: Prepare the reaction solution according to the reaction system and react at 30°C. Take samples at 2min, 4min, 6min, and 8min and add EDTA to a final concentration of 0.6M to terminate the reaction.

(5) Spotting: Take 1.2 μL of the reaction solution and spot it on the chromatography plate, spacing each sample 1cm apart.

(6) Expansion: Place the chromatography plate into a chromatography cylinder containing 25 mL of chromatography solution. When the chromatography solution expands to 1.5cm from the top, take out the chromatography plate and place it on clean filter paper to dry naturally. Dry or blow dry with a hair dryer.

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

(8) Process data: Use a Typhoon scanner to scan the phosphor screen to obtain the image, and then use ImageQuantTL to quantify the gray value and calculate the amount of hydrolysis.

The results are shown in Figure 2. 0μM PBL-peptide in Figure 2 represents the ATP hydrolysis of MBP FMDV 2C; 1.25μM PBL-peptide represents the ATP hydrolysis of MBP FMDV 2C in the presence of 1.25μM PBL-peptide; 12.5μM PBL -peptide represents ATP hydrolysis in the presence of 12.5μM PBL-peptide. As can be seen in Figure 2, PBL-peptide inhibits the ability of MBP FMDV 2C to hydrolyze ATP in a concentration-dependent manner.

Example 3: TAT-PBL-peptide membrane penetration efficiency detection

1. Preparation of TAT-PBL-peptide

Add the membrane-penetrating peptide TAT to the N-terminus of PBL-peptide, and name the new polypeptide TAT-PBL-peptide (N’-YGRKKRRQRRRNLHEKVASQPIFKQ-C’, as shown in sequence 1 in the sequence listing). In order to observe the membrane-penetrating effect of the polypeptide, a FITC label was added to the N-terminus of TAT-PBL-peptide and named: FITC-TAT-PBL-peptide.

2.TAT-PBL-peptide membrane penetration efficiency detection

Experimental method: Fluorescence microscopy observation

In this example, porcine kidney cells (PK-15 for short, purchased from Cell Resource Center, IBMS, CAMS/PUMC) were used as the research object. Prepare MEM complete medium (MEM medium was purchased from Gibco Company, in which 10% fetal calf serum and 100 mg/mL streptomycin were added). PK-15 cells were plated in a 12-well plate in advance. After the cells grew into a monolayer, MEM complete medium containing 5 μM FITC-TAT-PBL-peptide was added. After 12 hours, they were washed three times with PBS and examined under a fluorescence microscope and Observe the entry of peptides into cells under a confocal microscope. The results are shown in Figure 3. The left row of Mock in Figure 3 represents the cell fluorescence when no peptide is added, Brightfield represents the bright field situation, and FITC represents the dark field situation. FITC-TAT-PBL-peptide represents the fluorescence of cells in the presence of 5 μM FITC-TAT-PBL-peptide. Brightfield represents the bright field situation, FITC represents the dark field situation, and superomise represents the superposition of the two. The picture on the right shows the entry of polypeptide into cells observed under a confocal microscope. This experiment shows that TAT-PBL-peptide has good cell penetration and can penetrate the cell membrane. .

Example 4: PBL-peptide destroys intracellular aggregation of lipid droplets induced by FMDV 2C.

1.Construction method of N-mCherry-FMDV 2C plasmid

The sequence between NdeI and BamHI of plasmid pCDNA 3.1 (Thermo Fisher) was replaced with the encoding gene of mCherry and the FMDV 2C encoding gene. The FMDV 2C encoding gene sequence is as shown in GenBank ID: CAB62902.1, and the mCherry Coding genes such as GenBank ID: FM169983.1.

2. Experiment on PBL-peptide destroying intracellular aggregation of lipid droplets induced by FMDV 2C

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

(1) Plating: Plate PK-15 cells into a 12-well plate with cell sheets in advance, and wait until the cells grow into a monolayer the next day.

(2) Transfection: Use LipofectamineTM2000 (Invitrogen) as transfection reagent, 3 μL of transfection reagent per well, and 2 μg of N-mCherry-FMDV 2C plasmid. According to the instructions of the transfection reagent, mix the plasmid and the transfection reagent and let it stand for 20 minutes before adding it to the 12-well plate (change the MEM anti-serum-free medium in advance). After 6 hours, replace it with TAT containing 0 μM, 1 μM and 10 μM. -PBL-peptide's MEM complete medium MEM complete medium.

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

(4) Slide processing: Add 1.2 μL of anti-fluorescence quenching agent to the microscope slide in advance, and flip the cells onto the slide.

(5) Confocal microscope observation: Observe the slide through a confocal microscope.

The results are shown in Figure 4, (left picture: 2C localization and lipid droplet aggregation in the presence of 0 μM, 1 μM and 10 μM TAT-PBL-peptide respectively, mCherry represents the expression of 2C, Bodipy493/503 represents the expression of intracellular lipid droplets The situation, merge is the co-localization situation of the two, and zoom is the magnification of the white box in the Merge picture. The picture on the right shows the average number of lipid droplets in the cell when different concentrations of PBL-peptide exist.), it can be seen from Figure 4 that TAT-PBL- The peptide can destroy the intracellular lipid droplet aggregation induced by FMDV 2C. TAT-PBL-peptide can destroy the FMDV 2C-induced LD aggregation in PK-15 cells at low micromolar concentrations (1μM and 10μM).

Example 5: PBL-peptide cytotoxicity assay.

In the anti-viral process, the polypeptide PBL-peptide must not only inhibit the virus, but also ensure that it is non-toxic to cells. Therefore, this indicator was detected through a cytotoxicity test, and cells without any treatment were used as the control group.

Plate 96-well cell plate with PK-15 cells, 100 μL per well. When it reaches 70%-80% confluence, replace the MEM medium containing 10% serum with a medium containing a certain concentration gradient of the polypeptide PBL-peptide, so that the final PBL-peptide concentration in the well is 0.097 μM. , 0.19μM, 0.39μM, 0.78μM, 1.56μM, 3.125μM, 6.25μM, 12.5μM, 25μM, 50μM, 100μM, 200μM, 400μM, 800μM. 24 hours after adding the peptide, add 10 μL of viable cell detection agent CCK-8 to each well and mix well. Place at 37°C for 4 hours. Use a microplate reader to detect the absorbance value at OD450nm. The results are shown in Figure 5. Taking the viable cell amount of untreated cells as 100%, the viable cell amount after adding 100 μM polypeptide is basically consistent with the control group (untreated cells), proving that the CC ₅₀ of the polypeptide PBL-peptide is >800 μM.

Example 6: Determination of antiviral efficiency of PBL-peptide.

The FMDV O/BY/CHA/2010 strain used in the examples 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.

1. Experiment on cytopathic phenomena

PK-15 cells were seeded in a 96-well plate. After the cells grew into a monolayer, 100 μL FMDV (MOI=0.5) was added to each well. After incubation at 37°C for 2 hours, the virus liquid was discarded. PBL-peptide was added with MEM complete medium. The infected cells were diluted 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, and a normal cell control was established. 18 hours later, the cytopathic effects were observed through a microscope. The results are shown in Figure 6. The polypeptide PBL-peptide can inhibit the PK-15 cell pathology caused by FMDV, and the degree of inhibition is positively correlated with the concentration of the peptide.

2. Plaque formation experiment

PK-15 cells were inoculated into a 12-well plate. After 48 hours of culture, FMDV (MOI=0.01) was inoculated and incubated in a 37°C incubator for 1 hour. During this period, the culture plate was shaken once every 10 minutes. Then the virus liquid was discarded and added PBL-peptide was diluted with MEM complete medium so that the final PBL-peptide concentrations in each well were 0 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, 200 μM, and 400 μM. At the same time, a control of untreated normal cells was established. Incubate in a 37°C incubator for 4 hours, discard the supernatant, and perform a plaque experiment. The results are shown in Figure 7. The polypeptide PBL-peptide can reduce the number of plaques formed by PK-15 cells caused by FMDV replication, and the plaques are reduced. The extent is positively correlated with peptide concentration.

3. RT-qPCR determines viral RNA and calculates the 50% inhibitory concentration of the polypeptide (IC ₅₀ )

PK-15 cells were seeded in a 24-well plate. After the cells grew to a monolayer, they were infected with FMDV (MOI=0.5). After incubation at 37°C for 2 hours, the polypeptide diluted in MEM medium was added to make the final PBL- The peptide concentrations are 0 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, 200 μM, and 400 μM. At the same time, a normal cell control was established. 18 hours after infection, the supernatant was discarded, and total RNA was extracted from the infected cells. After reverse transcribing cDNA, the difference in FMDV replication levels was determined by fluorescence quantitative PCR. The GAPDH gene was used as an internal reference. According to the qPCR results, the IC ₅₀ was calculated using Graphpad Prism9 software to be 5.526 μM (as shown in Figure 8). This shows that the polypeptide PBL-peptide can effectively inhibit the replication of FMDV, and its IC ₅₀ is 5.526 μM.

In order to verify the specificity of PBL-peptides in inhibiting FMDV virus replication in cells and that it is not caused by the membrane-penetrating peptide TAT, the same method was used to detect the control polypeptide TAT-peptides (the amino acid sequence is 1-11 of sequence 1). The accumulation of viral RNA in infected cells after treatment, and the IC ₅₀ of the control polypeptide was calculated to be approximately 72 μM (as shown in Figure 9), which is much greater than TAT-PBL-peptide. Therefore, the effect of the membrane-penetrating peptide TAT on viral inhibition was ruled out.

4. Western Blot to determine viral protein expression

PK-15 cells were seeded in a 6-well plate and infected with FMDV (MOI=0.5). After 2 hours, the polypeptide diluted in MEM medium was added so that the final PBL-peptide concentrations in the wells were 0 μM, 3.125 μM, 6.25 μM, and 12.5μM, 25μM, 50μM, 100μM, 200μM, 400μM. At the same time, a normal cell control was established. Samples were collected 18 hours after infection, and the expression of viral VP1 protein was analyzed by Western Blot. The results showed that as the peptide concentration increased, the FMDV protein level showed a significant downward trend (Figure 10), indicating that the peptide PBL-peptide can inhibit the replication of FMDV.

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

Although the specific embodiments of the present invention have been described above in conjunction with the accompanying drawings, they do not limit the scope of the present invention. Those skilled in the art should understand that based on the technical solutions of the present invention, those skilled in the art do not need to perform creative work. Various modifications or variations that can be made are still within the protection scope of the present invention.

Industrial applications

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

Claims

A polypeptide whose amino acid sequence is shown in positions 12-25 of Sequence 1 of the sequence listing.
The derivative of the polypeptide of claim 1 is as follows (1) or (2) or (3):

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

(2) A derivative obtained by adding a functional peptide to one end of the polypeptide of claim 1;

(3) A derivative obtained by linking the polypeptide of claim 1 or the derivative of (1) to a carrier.
The derivative according to claim 2, characterized in that the functional peptide in (2) is a membrane-penetrating peptide.
The derivative according to claim 3, characterized in that the derivative in (2) is a polypeptide with an amino acid sequence as shown in Sequence 1 of the sequence listing.
Application of the polypeptide of claim 1 in the preparation of foot-and-mouth disease virus inhibitors or medicines for preventing foot-and-mouth disease virus.
A foot-and-mouth disease virus inhibitor whose active ingredient is the polypeptide described in claim 1 or the derivative described in any one of claims 2-4.
The use of the derivatives of any one of claims 2 to 4 in the preparation of medicines for treating or preventing foot-and-mouth disease virus or the preparation of foot-and-mouth disease virus inhibitors.
A medicine for treating or preventing foot-and-mouth disease virus, the active ingredient of which is the polypeptide described in claim 1 or the derivative described in any one of claims 2-4.
A method for treating or preventing foot-and-mouth disease virus, characterized by comprising the step of using the medicine for treating or preventing foot-and-mouth disease virus described in claim 8.