CN116999558B - Use of PAR1 as target for treating or inhibiting Ebola virus - Google Patents

Abstract

The invention discloses an application of PAR1 as a target spot for treating or inhibiting Ebola virus, belonging to the field of medical preparations. The invention discovers that PAR1 activation can promote the entry of virus GP protein into cells, and PAR1 knockout can inhibit the adsorption of EBOV virus-like particles on the surfaces of cells. Furthermore, it was found that knocking down or knocking out PAR1 significantly inhibited EBOV proliferation in cells, and over-expression of PAR1 could promote EBOV proliferation in cells. The present invention also found that PAR1 may be involved in EBOV adsorption and entry as a receptor or co-receptor. In addition, the invention proves that the PAR1 plays an important role in the proliferation of the EBOV, which proves that the PAR1 can be used as a candidate target point for the research and development of anti-EBOV medicines and has wide application prospect in the field of the treatment of the EBOV.

Description

Use of PAR1 as target for treating or inhibiting Ebola virus

Technical Field

The invention belongs to the field of medical preparations, and particularly relates to application of PAR1 as a target for treating or inhibiting Ebola virus.

Background

Ebola virus (eboov) belongs to the family of filoviridae and is a single-stranded negative-strand, non-segmented RNA virus containing an envelope. The virus can cause acute hemorrhagic infectious diseases of human beings and non-human primates, also called ebola virus disease (Ebola Virus Disease, EVD), and the death rate is about 25% -90%. The ebola epidemic in 2013-2016 caused 28000 cases of infection and 11000 cases of death worldwide. In 2018, ebola epidemic was again outbreaked in western africa, resulting in 3470 people being infected and 2287 people dying. In 2022, the Uganda outbreak of Ebola epidemic caused 141 cases of definite diagnosis and 55 cases of death. In order to ensure the health and safety of related personnel and reduce the EVD input risk, the reserve of prevention and treatment drugs aiming at the EBOV is very necessary.

The EBOV genome sequence is 3 '-end non-coding region-NP-VP 35-VP40-GP-VP30-VP24-L-5' -end non-coding region, and can code for 7 structural proteins including nucleoprotein NP, polymerase cofactor VP35, matrix protein VP40, glycoprotein GP, VP30, VP24 and RNA-dependent RNA polymerase L. Wherein the glycoprotein GP is the only protein located on the surface of the viral particle comprising two subunits GP1 and GP 2. GP1 is primarily involved in viral attachment to host cell receptors, while GP2 is primarily responsible for viral membrane fusion. EBOV infection of host cells is a complex multi-step process. The intracellular receptor NPC1 is now found to be the key intracellular receptor for EBOV to enter cells, and its interaction with GP is necessary for viral and cell membrane fusion. There are two main classes of receptors on the cell surface involved in viral adsorption and entry: c-type lectins (e.g., DC-SIGN and L-SIGN) and phosphatidylserine receptors (e.g., T-cell immunoglobulin mucins (TIMs)), the interaction of these cell surface protein receptors with EBOV virus particles is non-specific. Cell surface receptors capable of independently mediating EBOV entry have not been found.

Protease activated receptor 1 (PAR 1) is a 7-transmembrane G protein coupled receptor, which is activated by low doses of thrombin and is also known as a high affinity thrombin receptor. PAR1 is composed of 425 amino acids and is highly expressed on the surfaces of various cells such as endothelial cells, epithelial cells, immune cells, smooth muscle cells, platelets, neutrophils, macrophages and leukemia leukocytes. PAR1 consists of seven transmembrane domains (TM 1-7), one N-terminal domain containing a signal peptide, a Tethered Ligand (TL), three extracellular loops (ECL 1-3), three intracellular loops (ICL 1-3), an intracellular C-terminal domain and a C-tail helix 8 (H8). The mechanism of activation of PARs is very unique in G protein-coupled receptors. The N-terminus of PAR1 is cleaved by proteases, exposing the tethered ligand TL. TL is activated upon binding to its second extracellular loop ECL2, and this intramolecular activation mechanism results in conformational changes that are transmitted to G proteins, including gαq, gαi, gα13 and gγβ, and β -blockers, etc. It has now been found that various enzymes such as thrombin, tissue Factor (TF), MMP, etc., activate PAR1. Different activators trigger different signal paths after PAR1 is activated.

Disclosure of Invention

The invention aims to provide a target spot of PAR1 gene applied to ebola virus treatment and related products. Wherein the nucleotide sequence of the PAR1 gene is shown as a sequence 1, and the amino acid sequence of the PAR1 protein is shown as a sequence 2.

The invention claims the use of a substance that silences or knocks out or mutates the PAR1 gene or a substance that inhibits the expression of the PAR1 gene in any one of the following a 1) to a 8):

a1 Preparing a product for the treatment or adjuvant treatment of ebola virus disease;

a2 Screening for products that treat or adjunctively treat ebola virus disease;

a3 Preparing a product that inhibits ebola virus infection;

a4 Screening for products that inhibit ebola virus infection;

a5 Preparing a product for inhibiting ebola virus proliferation;

a6 Screening products for inhibition of ebola virus proliferation;

a7 Preparing a product for preventing ebola virus disease;

a8 Screening for products useful in the prevention of ebola virus disease.

The invention claims the use of substances which inhibit the activity of the PAR1 protein or of substances which reduce the content of the PAR1 protein in any of the following b 1) to b 8):

b1 Preparing a product for the treatment or adjuvant treatment of ebola virus disease;

b2 Developing or designing or screening products for the treatment or co-treatment of ebola virus disease;

b3 Preparing a product that inhibits ebola virus infection;

b4 Developing or designing or screening products that inhibit ebola virus infection;

b5 Preparing a product for inhibiting ebola virus proliferation;

b6 Developing or designing or screening a product that inhibits ebola virus proliferation;

b7 Preparing a product for preventing ebola virus disease;

b8 Development or design or screening of products for prevention of ebola virus disease.

The invention relates to a product whose active ingredient is a substance which inhibits the activity of the PAR1 protein or which reduces the content of the PAR1 protein or which silences or knocks out or mutates the PAR1 gene or which inhibits the expression of the PAR1 gene.

The above substance inhibiting PAR1 protein activity or substance reducing PAR1 protein content is protein, polypeptide or small molecule compound which inhibits PAR1 protein synthesis or promotes PAR1 protein degradation or inhibits PAR1 protein function.

The substance for inhibiting PAR1 gene expression is siRNA for inhibiting PAR1 gene expression; the siRNA for inhibiting PAR1 gene expression is siRNA formed by annealing two single strands shown in a sequence 3 and a sequence 4 or siRNA formed by annealing two single strands shown in a sequence 5 and a sequence 6.

The substance for knocking out the PAR1 gene is a CRISPR/Cas9 gene editing system for knocking out the PAR1 gene;

the sgRNA target sequence in the CRISPR/Cas9 gene editing system for knocking out the PAR1 gene is a sequence 7 or a sequence 8.

The invention also claims the use of PAR1 as a target in any of the following c 1) to c 8):

c1 Preparing a product for the treatment or adjuvant treatment of ebola virus disease;

c2 Developing or designing or screening products for the treatment or co-treatment of ebola virus disease;

c3 Preparing a product that inhibits ebola virus infection;

c4 Developing or designing or screening products that inhibit ebola virus infection;

c5 Preparing a product for inhibiting ebola virus proliferation;

c6 Developing or designing or screening a product that inhibits ebola virus proliferation;

c7 Preparing a product for preventing ebola virus disease;

c8 Development or design or screening of products for prevention of ebola virus disease.

The invention also claims the use of PAR1 for interaction with ebola virus protein GP.

The invention also claims any of the following biomaterials d 1) -d 3):

d1 An siRNA formed by annealing two single strands shown in sequence 3 and sequence 4 or an siRNA formed by annealing two single strands shown in sequence 5 and sequence 6;

d2 A sgRNA whose target sequence is sequence 7 or sequence 8;

d3 PAR1 gene editing system comprising Cas9 protein and d 2) the sgRNA.

The use of the above-described biomaterials in any one of the following e 1) to e 8) is also within the scope of the present invention:

e1 Preparing a product for the treatment or adjuvant treatment of ebola virus disease;

e2 Developing or designing or screening products for the treatment or co-treatment of ebola virus disease;

e3 Preparing a product that inhibits ebola virus infection;

e4 Developing or designing or screening products that inhibit ebola virus infection;

e5 Preparing a product for inhibiting ebola virus proliferation;

e6 Developing or designing or screening a product that inhibits ebola virus proliferation;

e7 Preparing a product for preventing ebola virus disease;

e8 Development or design or screening of products for prevention of ebola virus disease.

The present invention utilizes immunoprecipitation and immunofluorescence to confirm the intracellular interaction and co-localization of GP and PAR1. PAR1 activation can promote GP entry into cells, PAR1 knockout can inhibit EBOV adsorption on cell surfaces. Furthermore, EBOV trVLP infection can recruit PAR1 to viral inclusion bodies and thereby reduce PAR1 distribution on the cell surface. Knocking down or knocking out PAR1 significantly inhibits EBOV proliferation in cells and EBOV vRNA levels in cells and culture supernatants, and over-expression of PAR1 can promote EBOV proliferation in cells.

The invention discovers that the interaction of the EBOV GP and the PAR1 promotes the virus to enter cells and the internalization of the PAR1, and the knockout of the PAR1 inhibits the adsorption of the EBOV tr VLP on the cell surface, which indicates that the PAR1 possibly serves as a receptor or an auxiliary receptor to participate in the adsorption and the entry of the EBOV. Experiments such as PAR1 knocking down, knocking out, reverting and over-expression prove that PAR1 plays an important role in EBOV proliferation, and the PAR1 can be used as a candidate target point for the research and development of anti-EBOV medicines, and has important application value for the treatment of ebola virus diseases.

Drawings

FIG. 1 shows the interaction and co-localization of GP with PAR1, wherein A is the interaction between GP and PAR1 detected by immunoprecipitation, and B is the co-localization of GP and PAR1 detected by immunofluorescence in cells.

FIG. 2 is a graph showing the effect of PAR1 activation on GP entry cells, wherein A is the effect of PAR1 activation on GP entry cells, B is the effect of qRT-PCR detection on PAR1 knockout on EBOV adsorption, and C is the PCR result graph after PAR1 knockout.

FIG. 3 shows that EBOV trVLP infection promotes PAR1 internalization; wherein control represents control untransfected sample, trVLP represents EBOV virus-like particles, and MFI represents mean fluorescence intensity.

FIG. 4 shows that EBOV trVLP infection recruits PAR1 into viral inclusion bodies.

FIG. 5 shows that knockout of PAR1 inhibits proliferation of EBOV tr VLPs in cells; wherein A is qRT-PCR for detecting PAR1 knockdown efficiency in HepG2 cells; b is the inhibition of EBOV trVLP proliferation in HepG2 cells by PAR1 knockdown; c is the effect of immunofluorescence detection PAR1 knockdown on proliferation of EBOV tr VLPs in HepG2 cells; d is qRT-PCR to detect PAR1 knock-down efficiency in HuH7 cells; e is the inhibition of proliferation of EBOV tr VLPs in HuH7 cells by PAR1 knockdown.

FIG. 6 shows that knockout PAR1 inhibits proliferation of EBOV tr VLPs in cells and culture supernatants.

FIG. 7 shows that overexpression of PAR1 promotes proliferation of EBOV tr VLPs in cells.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified. The quantitative tests in the following examples were all set up in triplicate and the results averaged.

The cell lines and plasmids referred to in the examples below: HEK293 cells and HuH7 cells are purchased from the cell resource center of the basic medical institute of China medical sciences; hepG2 cells were purchased from source well organisms; plasmids Myc-PAR1 (HG 13535-NM) and GP-GFP (VG 40304-ACG), flag-GP (VG 40304-NF) were purchased from Sino Biological; flag-GP1 and Flag-GP2 are synthesized in general biology; pCAGGS-V, pGL3-Promoter purchased from Ubbelopsis; pCAGGS-NP, pCAGGS-VP35, pCAGGS-VP30, pCAGGS-L, pCAGGS-T7, pCAGGS-Tim1 and p4cis-vRNA-Luc are described in the literature Hoenen T, et al J. Vis. Exp., 2014). Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes, which are available to the public from the military medical institute for the purpose of repeating the experiment.

The molecular biological reagents and antibodies referred to in the examples below: high-fidelity DNA polymerase KOD FX Neo, SYBR Green Mix purchased from Toyobo, FSQ 301; cDNA reverse Mix (R333-01) was purchased from Vazyme; the double luciferase assay kit was purchased from (E640A) Promega; transfection reagent Lipofectamine3000 (L3000-015) was purchased from Thermo company; protease inhibitor Cocktail is purchased from roche company (04693132001); DMEM medium (C11995500 BT), MEM medium (C11095500 BT), NEAA (11140-500) were purchased from GIBCO. Fetal bovine serum (FSP 500) was purchased from ExCell biologies. HRP-labeled anti-Flag antibody (A8592-1 MG) and HRP-labeled anti-Myc antibody (SAB 4200742-1 VL) were purchased from Sigma company; anti-PAR1 (PA 5-116040) antibodies are purchased from Invitrogen corporation; anti-Flag agarose beads (A2220-1 ML) and anti-Myc agarose beads (E6654-1 ML) were purchased from Sigma.

The nucleotide sequence corresponding to the PAR1 gene (GeneBank: NM-001992.5, updated on day 29 of 2023) in the following examples is shown in SEQ ID NO. 1, and the amino acid sequence of the encoded PAR1 protein is shown in SEQ ID NO. 2.

Example 1 immunoprecipitation detection of GP and PAR1 interactions in cells.

Using 60mm dishes of 293 cells co-transfected with Myc-PAR1 and GP-GFP plasmids as an example, transfection was performed according to the Thermo company Lipofectamine3000 instructions, briefly as follows: mu.g of Myc-PAR1 and 2. Mu.g of GP-GFP plasmid and 8. Mu.l of P3000 were diluted with 100. Mu.l of opti-MEM; mu.l of Lipofectamine3000 was diluted with 100. Mu.l of opti-MEM, and the diluted plasmid and P3000 mixture was added dropwise to the diluted Lipofectamine3000 and mixed well; the plasmid-liposome mixture was added dropwise to the cell culture medium after 15min at room temperature.

After 36-48h of transfection, the cells are resuspended and washed for 2 times by PBS, and the cells are collected by centrifugation at 1000g/min for 3min at 4 ℃; adding 400 μl of cell lysate (150mM NaCl,50mM Tris-HCl pH8.0, 1 tablet/50 ml containing EDTA protease inhibitor, 1% NP 40) on ice, lysing for 20min, centrifuging at 4deg.C at 1,2000rpm for 10min; the supernatant was transferred to a 1.5ml EP tube, 15. Mu.l of anti-GFP antibody coupled to agarose beads was added, and the mixture was incubated at 4℃for 2h for co-immunoprecipitation, centrifuged at 1000g for 3min at 4℃and the cells were washed 3 times with 1000. Mu.l of cell lysate without protease inhibitor; adding proper amount of 1 XSDS loading buffer, boiling water for 8min, centrifuging at 4 ℃ and 16000g/min for 5min, and performing SDS-PAGE electrophoresis and immunoblotting.

Taking 10 μl of sample for SDS-PAGE electrophoresis, setting the voltage at 80V initially, adjusting the voltage to 120V after each strip of the Marker is obviously separated, continuing electrophoresis until bromophenol blue migrates to the bottom of the gel, and stopping electrophoresis; PVDF membrane was activated with methanol for 30s and then soaked with filter paper in 1 Xtransfer buffer (Tris-HCl 24mM, glycine 5mM,20% methanol) for 30min; after electrophoresis, placing the filter paper, the glue, the membrane and the filter paper on a semi-dry membrane transferring instrument in sequence from top to bottom, and transferring the membrane for 2 hours at 18V; then sealing the PVDF film for 1h at room temperature; washing with 1 XTBST for 3 times and 5min each time (washing for short); adding Myc-HRP and GFP-HRP, and incubating for 1h at room temperature; ECL development was performed after TBST washing.

The results are shown as a in fig. 1, and the interaction between EBOV GP and PAR1 in cells was confirmed by immunoprecipitation experiments.

Example 2 immunofluorescence detection GP and PAR1 co-localize in cells.

1 μg Myc-PAR1 and 1 μg GP-GFP or GFP were transfected in HepG2 cells using Lipofectamine3000 from Thermo company (procedure shown in example 1). Washing with PBS 3 times after 48h transfection to absorb residual liquid, adding 4% paraformaldehyde, and fixing at 37deg.C for 30min; after PBS washing, 0.3% Triton X-100 (1 XPBS) was added and perforated for 15min; adding 2ml of blocking solution (1 XPBS containing 5% goat serum) at 37deg.C for 30min; after PBS washing, anti-PAR1 antibodies (diluted 1:50 in blocking solution) are added, and the mixture is incubated at 4 ℃ overnight or at room temperature for 1h; cells were washed 3 times for 10min each with 1 XPBST (abbreviated PBST wash); adding TRITC-labeled goat anti-rabbit antibody (diluted 1:00 of blocking solution) into the cells, and incubating for 1h at room temperature in a dark place; after PBST washing, 10. Mu.l of a DAPI (1. Mu.g/ml) containing blocking solution was added, and after standing for 15min, the distribution of PAR1 and GFP in cells was examined by a laser confocal microscope (Carl Zeiss LSM 800).

As a result, as shown in FIG. 1B, it was found that GP co-localized with PAR1 on the surface of cell membrane by immunofluorescence technique.

Example 3 immunofluorescence detects the effect of PAR1 activation on GP entry into cells.

1. Mu.g of GP-GFP or GFP was transfected into HepG2 cells using Lipofectamine3000 from Thermo company (procedure shown in example 1). PAR1 agonistic peptide (AP, TFLLRN) was added at 50. Mu. Mol/L for 10min after 48h of transfection, followed by washing and fixing of cells, perforation, blocking, incubation of PAR1 antibody and TRITC fluorescent secondary antibody (procedure as shown in example 2), and detection of the effect of activated PAR1 on GFP distribution in cells.

The results are shown in FIG. 2, A, and show that, following agonist-activated internalization of PAR1 into cells, GP located on the surface of the cell membrane also enters cells and co-localizes with internalized PAR1.

Example 4, qRT-PCR examined the effect of PAR1 knockout on cell surface adsorption of EBOV trvlps.

In order to verify the effect of PAR1 on the cell surface adsorption of EBOV trVLP, a HepG2 cell line for knocking out 299bp of PAR1 gene is constructed by using CRISPR/Cas9 technology (the knocking-out sequence is shown as 53-351 bits of sequence 9 in a sequence table), and the brief flow is as follows: (1) sgRNA construction: designing two pairs of sgrnas targeting PAR1 (sgrnas 1: AAATGACCGGGGATCTAAGGTGG shown as sequence 7 in a sequence table; sgrnas 2: TGCAGCATGTACACCACCGCCGG shown as sequence 8 in the sequence table) by utilizing an sgRNA sequence online design website (http:// crispr. Mit. Edu /), adding a sticky end CACCG at the 5 'end of a sense strand of the sgrnas, and adding a sticky end AAAC at the 5' end of an antisense strand; (2) The synthesized sgRNA sequence (beijing noxel genome research center limited) was annealed and cloned into the pSpCas9 (BB) -2A-Puro (PX 459) vector (methods reference molecular cloning experiments (third edition) guidelines). (3) Co-transfecting the two pairs of recombinant plasmids into HepG2 cells, and screening out monoclonal antibodies by using 1 mug/ml puromycin after 48h of transfection; (4) Limiting dilution of the obtained clone to 96 well plates, and enlarging culture to 24 well plates and 6 well plates; (5) screening positive clonal cell lines: designing an upstream primer and a downstream primer aiming at the sgRNA targeting sequence, and amplifying upstream and downstream fragments with target gene fragments; agarose gel electrophoresis detection and sequencing confirmation, and screening out PAR1 knockout cell lines.

Designing a primer F1: GGGTAGATCTCTGAAAACCTATC on the upstream of the sgRNA1 according to the nucleic acid sequence, as shown in a sequence 10 in a sequence table; the primer R1: GATGTTGAGCCCGGGCACC is designed at the downstream of the sgRNA2 and is shown as a sequence 11 in a sequence table; primer R2: CTGACTACAAACACTCCGGTG is designed between the gRNA1 and the gRNA2, and is shown as a sequence 12 in a sequence table; designing two pairs of F1/R1 and F1/R2 primers for PCR amplification to verify that the knockout was successful, wherein the PCR result is shown as C in FIG. 2, the nucleic acid electrophoresis size of the amplified wild type cells (WT) is 933bp (lane 1), the nucleic acid electrophoresis size of the amplified mutant cells (#D12) is 634bp (lane 2), the nucleic acid electrophoresis size of the amplified wild type cells is 527bp (lane 3), and the nucleic acid electrophoresis size of the amplified mutant cells is 0bp (lane 4); the results indicate that PAR1 gene knockout was successful.

EBOV minimal genome system-related plasmids were transfected in HepG2 cells, respectively: pCAGGS-NP (125 ng), pCAGGS-VP35 (125 ng), pCAGGS-VP30 (75 ng), pCAGGS-L (1000 ng), p4cis-vRNA-Rluc (250 ng) and pCAGGS-T7 (250 ng) (the procedure is as in example 1). Cell supernatants were collected 48h of transfection to obtain ebola virus-like particles (trvlps). EBOV trvlps not adsorbed in the cells were washed with pre-chilled PBS after adsorption for 1h at 4 ℃ with addition of EBOV trVLP supernatant to HepG2 cells and PAR1 knockdown cells. The cells were collected and viral genomic RNA was extracted in a biosafety cabinet according to the instructions QIAamp Viral RNA Mini Kit (Qiagen) (52906). cDNA sequence (totol RNA 1. Mu.g, 5×all-in-one qRT-PCR Supermix 4. Mu.l, enzyme Mix 1. Mu.l, ddH) was then reverse transcribed according to the Noruzan reverse transcription kit (R333-01) ₂ O to 20. Mu.l; the procedure was 50℃for 15min;85 ℃,5 s). Detecting RNA transcription level of VP40 in cells by Quantum studio6 fluorescence quantitative PCR (VP 40F: GGAGGCCATATACCCTGTCAGGTC, shown as sequence 13 in the sequence table, VP40R: GCCTGGTGTGTGGCTGGCAT, shown as sequence 14 in the sequence table, GAPDH-F: AAGGTCATCCCTGAGCTGAAC, shown as sequence table) by SYBR Green Mix (Toyobo) methodSequence 15; GAPDH-R: ACGCCTGCTTCACCACCTTCT as shown in sequence 16 in the sequence listing).

The results are shown in fig. 2B, which shows that PAR1 knockout can significantly inhibit the adsorption capacity of EBOV on the cell surface.

Example 5, flow cytometry examined the effect of EBOV tr vlp infection on PAR1 distribution on the cell surface.

Transfection of Myc-PAR1 500ng and EBOV minimal genome System-related plasmids in HepG2 cells: pCAGGS-NP (125 ng), pCAGGS-VP35 (125 ng), pCAGGS-VP30 (75 ng), pCAGGS-L (1000 ng), p4cis-vRNA-Rluc (250 ng) and pCAGGS-T7 (250 ng) (the procedure is as in example 1). Cells were collected 48h after transfection, washed 2 times with 1 XPBS, incubated with Myc antibody (1:50 dilution) for 1h, followed by FITC fluorescent secondary antibody, and the effect of EBOV trVLP infection on PAR1 distribution on membrane surface was analyzed statistically by flow cytometry (Millipore ImageStreamX MarkII).

As shown in fig. 3, it was found by flow cytometry that EBOV tr vlp infection significantly reduced the distribution of PAR1 on the cell membrane surface in HepG 2.

Example 6 immunofluorescence detection of effects of EBOV trVLP infection at different times on PAR1 distribution in cells.

Transfection of EBOV minimal genome system-related plasmids in HepG2 cells: pCAGGS-NP (125 ng), pCAGGS-VP35 (125 ng), pCAGGS-VP30 (75 ng), pCAGGS-L (1000 ng), p4cis-vRNA-Rluc (250 ng) and pCAGGS-T7 (250 ng) (the procedure is as in example 1). Cells were harvested after transfection for 0h, 6h, 12h, 18h, 24h, 36h and 48h, respectively. The effect of EBOV infection on PAR1 distribution in cells was then examined by washing and fixing cells, perforating, blocking, incubating PAR1 and VP35 antibodies, and FITC and TRITC fluorescent secondary antibodies (procedure shown in example 2).

As a result, as shown in FIG. 4, EBOV trVLPs at various time points (0 h, 6h, 12h, 18h, 24h, 36h and 48 h) were infected in HepG2 cells, and the dynamic change of PAR1 distribution in the cells was detected. The results showed that PAR1 was gradually recruited to the viral replication site, viral inclusion bodies, 18h after EBOV infection.

Example 7 effect of PAR1 knockdown on proliferation of EBOV tr vlps in cells.

EBOV minimal genome system: the present study utilizes an EBOV minimal genome system operable in a biosafety secondary laboratory to detect viral levels in cells by luciferase activity. The experimental operation flow is briefly as follows: on day 1, virus-producing cells (p 0 for short) were plated at 3X10 per well ⁵ Inoculating in 6-well plate at 37deg.C with 5% CO ₂ Is subjected to stationary culture in an incubator; on day 2, plasmids pCAGGS-NP (125 ng), pCAGGS-VP35 (125 ng), pCAGGS-VP30 (75 ng), pCAGGS-L (1000 ng), p4cis-vRNA-Rluc (250 ng) and pCAGGS-T7 (250 ng) were transfected into p0 cells according to lipofectamine3000 instructions; on day 3, the p0 supernatant was replaced with fresh medium containing 5% fbs. On day 4, virus-targeted cells (p 1 for short) were 3X10 per well ⁵ Individual inoculations were performed in 6-well plates; on day 5, plasmids pCAGGS-NP (125 ng), pCAGGS-VP35 (125 ng), pCAGGS-VP30 (75 ng), pCAGGS-L (1000 ng) and pCAGGS-Tim1 (250 ng) were transfected into p1 cells according to lipofectamine3000 instructions; on day 6, p0 cell supernatants were collected and replication of the minimal genome of ebola virus in p0 cells was detected using a dual luciferase assay kit. Replacing the supernatant of p1 cells with the cell supernatant of p 0; on day 7, the p1 supernatant was changed to medium of 5% FBS, and p1 was collected after further culturing for 72 hours. If the virus needs to be continuously passaged to obtain p2, the method is consistent with the p1 acquisition flow.

Luciferase activity assay: PAR1 siRNA (sense: 5'-GGCUACUAUGCCUACUACUTT-3', as shown by the T-turn U in SEQ ID NO: 3; anti: AGUAGUAGGCAUAGUAGCCUU-3', as shown by the T-turn U in SEQ ID NO: 4) or Scr siRNA (sense, 5'-UUCUCCGAACGUGUCACGUTT-3', as shown by the T-turn U in SEQ ID NO: 5; anti: 5'-ACGUGACACGUUCGGAGAATT-3', as shown by the T-turn U in SEQ ID NO: 6) was transfected with Thermo Lipofectamine3000 in HepG2 or HuH7 cells, and then transfected with the EBOV minimal genome system-related plasmid and firefly reporter pGL3-Basic (25 ng) after 6h of transfection. Collecting p0 cells in the minimum genome system, washing with PBS, adding 500 μl PLB lysate, lysing for 15min on a shaker, centrifuging, adding 20 μl cell lysis supernatant into 100 μ l Luciferase Assay Reagent II, mixing, adding into a TD-20/20 fluorescence photometer to determine luminescence value RLU1, and adding 100 μl Stop & Glo Reagent to determine fluorescence luminescence value RLU2. The relative luciferase activity values of RLU2/RLU1 assess the viral levels, confirming the effect of PAR1 knockdown on EBOV tr vlp proliferation in the cells.

Immunofluorescence detection: PAR1 siRNA or Scr siRNA was transfected with Lipofectamine3000 from Thermo company in HepG2 or HuH7 cells, and EBOV minimal genome system-related plasmids were transfected after 6h of transfection, cells were collected after 48h of transfection, followed by washing and fixing of cells, perforation, blocking, incubation of PAR1 and VP35 antibodies, and FITC and TRITC fluorescent secondary antibodies (procedure as in example 2), and the effect of PAR1 knockdown on EBOV tr VLP proliferation was analyzed by VP35 expression.

Results as shown in fig. 5, the present example uses the EBOV minimal genome system to detect the effect of PAR1 knockdown and knockdown on EBOV trVLP proliferation by luciferase activity assay. The PAR1 siRNA knockdown endogenous PAR1 in HepG2 cells (as shown by a in fig. 5) was transfected into HepG2 cells, followed by the EBOV minimal genome related plasmid, and luciferase activity experiments found that knocking down PAR1 significantly inhibited EBOV tr vlp proliferation (-2.7 fold) in cells (as shown by B in fig. 5). Immunofluorescence experiments can intuitively find that PAR1 knockdown significantly inhibited the proliferation of EBOV tr vlps in cells (indicated by VP 35) (as shown by C in fig. 5). Second, knocking down PAR1 in HuH7 cells also significantly inhibited EBOV trVLP proliferation (-3.0 fold) (as shown by D in fig. 5 and E in fig. 5).

Example 8 effect of PAR1 knockout on proliferation of EBOV tr vlps in cells.

Luciferase activity assay: EBOV minimal genome-related plasmid and firefly reporter pGL3-Basic (25 ng) were transfected in HepG2 and PAR1 knockout (PAR 1 KO) cells. The dual luciferase activity assay was identical to that described above (consistent with the luciferase activity assay described in example 7) and the effect of PAR1 knockout on EBOV tr vlp proliferation in cells was analyzed.

qRT-PCR detection: EBOV minimal genome-related plasmids were transfected into HepG2 and PAR1 knockout (PAR 1 KO) cells, the cells and culture supernatants were collected 120h after transfection, RNA was extracted, and qRT-PCR detection was performed after inversion (the method was identical to that of example 4), and the effect of PAR1 knockout on EBOV RNA in cells and cell supernatants was analyzed.

As shown in fig. 6, the effect of PAR1 knockout on EBOV trVLP proliferation was examined in this example, and the minimal genome related plasmid of EBOV was transfected in HepG2 and PAR1 knockout cells, and the luciferase results showed: EBOV tr vlp proliferation was reduced by about 2-fold and 12-fold in p 0-and p 1-generation cells after PAR1 knockout compared to HepG2 wild-type cells (as shown by a in fig. 6). When Myc-PAR1 and EBOV minimal genome-related plasmids were transfected in PAR1 knockout cells, the results showed that reversion of PAR1 expression in PAR1 knockout cells could significantly promote proliferation of EBOV trvlps (as shown by B in fig. 6). Furthermore, PAR1 knockdown was found by qRT-PCR experiments to significantly inhibit EBOV RNA levels (-12-fold and-16-fold) in cells and culture supernatants (as shown by C in fig. 6 and D in fig. 6).

Example 9 effect of PAR1 overexpression on proliferation of EBOV tr vlps in cells.

Luciferase activity assay: myc-PAR1 (or Myc) was transfected in HepG2 cells, and EBOV minimal genome system-related plasmid and firefly reporter pGL3-Basic (25 ng) were transfected after 6h (the procedure was the same as in example 7) to analyze the effect of PAR1 overexpression on EBOV trVLP proliferation in cells.

As shown in FIG. 7, overexpression of PAR1 can promote proliferation of EBOV trVLPs in cells (-3.0 fold).

In combination with the results of the above examples, the present invention found that EBOV GP interaction with PAR1 promotes viral entry into cells and PAR1 internalization, and that knockout of PAR1 inhibits EBOV tr vlp adsorption on cell surfaces, suggesting that PAR1 may be involved in EBOV adsorption and entry as a receptor or co-receptor. Experiments such as PAR1 knocking down, knocking out, reverting and over-expression prove that PAR1 plays an important role in EBOV proliferation, and the PAR1 can be used as a candidate target point for the research and development of anti-EBOV medicines, and has important application value for the treatment of ebola virus diseases.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Claims (2)

1. Use of a substance which knocks out the PAR1 gene or a substance which inhibits the expression of the PAR1 gene in any one of the following a 1) to a 4), wherein the nucleotide sequence of the PAR1 gene is as shown in sequence 1,

the substance for inhibiting PAR1 gene expression is siRNA formed by annealing two single strands with sequences GGCUACUAUGCCUACUACUTT and AGUAGUAGGCAUAGUAGCCUU;

the substance for knocking out the PAR1 gene is a CRISPR/Cas9 gene editing system for knocking out the PAR1 gene;

the sgRNA target sequence in the CRISPR/Cas9 gene editing system for knocking out the PAR1 gene is a sequence 7 or a sequence 8:

a1 Preparing a product for the treatment or adjuvant treatment of ebola virus disease;

a2 Preparing a product that inhibits ebola virus infection;

a3 Preparing a product for inhibiting ebola virus proliferation;

a4 A product for preventing ebola virus disease.

2. Use of a substance that inhibits PAR1 protein activity or a substance that reduces PAR1 protein content in any of the following b 1) -b 4), wherein the amino acid sequence of PAR1 protein is as shown in sequence 2, and wherein the substance that inhibits PAR1 protein activity or the substance that reduces PAR1 protein content is an siRNA formed by annealing two single strands of sequences GGCUACUAUGCCUACUACUTT and AGUAGUAGGCAUAGUAGCCUU:

b1 Preparing a product for the treatment or adjuvant treatment of ebola virus disease;

b2 Preparing a product that inhibits ebola virus infection;

b3 Preparing a product for inhibiting ebola virus proliferation;

b4 A product for preventing ebola virus disease.