CN116999558B - Application of PAR1 as a 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

Application of PAR1 as a target for treating or inhibiting Ebola virus

Technical field

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

Background technique

Ebola virus (EBOV) belongs to the family Filoviridae and is an enveloped single-stranded negative-sense non-segmented RNA virus. The virus can cause an acute hemorrhagic infectious disease in humans and non-human primates, also known as Ebola Virus Disease (EVD), with a mortality rate of approximately 25%-90%. The Ebola epidemic from 2013 to 2016 caused 28,000 infections and 11,000 deaths worldwide. In 2018, the Ebola epidemic broke out again in West Africa, causing 3,470 infections and 2,287 deaths. In 2022, an Ebola epidemic broke out in Uganda, resulting in a total of 141 confirmed cases and 55 deaths. In order to protect the health and safety of relevant personnel and reduce the risk of EVD import, it is necessary to reserve prevention and treatment drugs for EBOV.

The sequence of the EBOV genome is 3' non-coding region-NP-VP35-VP40-GP-VP30-VP24-L-5' non-coding region, which can encode nuclear protein NP, polymerase cofactor VP35, matrix protein VP40, and glycoprotein GP, VP30, VP24, and RNA-dependent RNA polymerase L, a total of 7 structural proteins. Among them, glycoprotein GP is the only protein located on the surface of viral particles and contains two subunits, GP1 and GP2. GP1 is mainly involved in the attachment of viruses to host cell receptors, while GP2 is mainly responsible for viral membrane fusion. EBOV infection of host cells is a complex multi-step process. Current research has found that the intracellular receptor NPC1 is the key intracellular receptor for EBOV to enter cells, and its interaction with GP is necessary for the fusion of the virus and the cell membrane. There are two main types of receptors involved in virus adsorption and entry on the cell surface: C-type lectins (such as DC-SIGN and L-SIGN) and phosphatidylserine receptors (such as T-cell immunoglobulin mucin (TIM) ), the interaction of these cell surface protein receptors with EBOV viral particles is nonspecific. No cell surface receptor that can independently mediate EBOV entry has been found.

Protease-activated receptor 1 (PAR1) is a 7-transmembrane G protein-coupled receptor. Because it can be activated by low doses of thrombin, it is also called a high-affinity thrombin receptor. PAR1 consists of 425 amino acids and is highly expressed on the surface of endothelial cells, epithelial cells, immune cells, smooth muscle cells, platelets, neutrophils, macrophages and leukemic leukocytes. PAR1 consists of seven transmembrane domains (TM1-7), an N-terminal domain containing a signal peptide, a tethering ligand (TL), three extracellular loops (ECL1-3), and three intracellular loops ( ICL1-3), the intracellular C-terminal domain and the C-tail helix 8 (H8). The activation mechanism of PARs is very unique among G protein-coupled receptors. The N terminus of PAR1 is cleaved by proteases, exposing the tethered ligand TL. TL is activated after binding to its second extracellular loop, ECL2. This intramolecular activation mechanism leads to conformational changes that are transmitted to G proteins, including Gαq, Gαi, Gα13 and Gγβ, and β-blockers. It has been found that thrombin, tissue factor (TF), MMP and other enzymes can activate PAR1. Different activators trigger different signaling pathways after activating PAR1.

Contents of the invention

The purpose of the present invention is to provide a target and related products for the application of PAR1 gene in the treatment of Ebola virus. Among them, the nucleotide sequence of the PAR1 gene is shown in Sequence 1, and the amino acid sequence of the PAR1 protein is shown in Sequence 2.

The present invention requires the use of substances that silence, knock out or mutate the PAR1 gene or substances that inhibit the expression of the PAR1 gene in any of the following a1)-a8):

a1) Prepare products for the treatment or auxiliary treatment of Ebola virus disease;

a2) Screen products for the treatment or adjuvant treatment of Ebola virus disease;

a3) Prepare products that inhibit Ebola virus infection;

a4) Screen products that inhibit Ebola virus infection;

a5) Prepare products that inhibit the proliferation of Ebola virus;

a6) Screen products that inhibit the proliferation of Ebola virus;

a7) Preparation of products for the prevention of Ebola virus disease;

a8) Screen products for the prevention of Ebola virus disease.

The present invention requires the use of substances that inhibit PAR1 protein activity or substances that reduce PAR1 protein content in any of the following b1)-b8):

b1) Preparation of products for the treatment or auxiliary treatment of Ebola virus disease;

b2) Develop or design or screen products for the treatment or adjuvant treatment of Ebola virus disease;

b3) Prepare products that inhibit Ebola virus infection;

b4) Develop or design or screen products that inhibit Ebola virus infection;

b5) Prepare products that inhibit the proliferation of Ebola virus;

b6) Develop or design or screen products that inhibit the proliferation of Ebola virus;

b7) Preparation of products for the prevention of Ebola virus disease;

b8) Develop or design or screen products for the prevention of Ebola virus disease.

The present invention claims a product whose active ingredient is a substance that inhibits the activity of PAR1 protein, a substance that reduces the content of PAR1 protein, a substance that silences, knocks out or mutates the PAR1 gene, or a substance that inhibits the expression of the PAR1 gene.

The above-mentioned substances that inhibit PAR1 protein activity or reduce PAR1 protein content are proteins, polypeptides or small molecule compounds that inhibit PAR1 protein synthesis or promote PAR1 protein degradation or inhibit PAR1 protein function.

The above-mentioned substance that inhibits the expression of the PAR1 gene is siRNA that inhibits the expression of the PAR1 gene; the siRNA that inhibits the expression of the PAR1 gene is an siRNA formed by the annealing of two single strands shown in Sequence 3 and Sequence 4 or a siRNA shown in Sequence 5 and Sequence 6 siRNA formed by the annealing of two single strands.

The above-mentioned material that knocks out the PAR1 gene is the CRISPR/Cas9 gene editing system that knocks out the PAR1 gene;

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

The present invention also claims the use of PAR1 as a target in any of the following c1)-c8):

c1) Prepare products for the treatment or auxiliary treatment of Ebola virus disease;

c2) Develop or design or screen products for the treatment or adjuvant treatment of Ebola virus disease;

c3) Prepare products that inhibit Ebola virus infection;

c4) Develop or design or screen products that inhibit Ebola virus infection;

c5) Prepare products that inhibit the proliferation of Ebola virus;

c6) Develop or design or screen products that inhibit the proliferation of Ebola virus;

c7) Preparation of products for the prevention of Ebola virus disease;

c8) Develop or design or screen products for the prevention of Ebola virus disease.

The present invention also claims the use of PAR1 in interacting with Ebola virus protein GP.

The present invention also claims protection for any biological material from d1) to d3) below:

d1) A siRNA, which is an siRNA formed by the annealing of two single strands shown in Sequence 3 and Sequence 4 or an siRNA formed by the annealing of two single strands shown in Sequence 5 and Sequence 6;

d2) An sgRNA whose target sequence is sequence 7 or sequence 8;

d3) PAR1 gene editing system, which includes Cas9 protein and the sgRNA described in d2).

The application of the above-mentioned biological materials in any of the following e1)-e8) should also be within the protection scope of the present invention:

e1) Preparation of products for the treatment or auxiliary treatment of Ebola virus disease;

e2) Develop or design or screen products for the treatment or adjuvant treatment of Ebola virus disease;

e3) Prepare products that inhibit Ebola virus infection;

e4) Develop or design or screen products that inhibit Ebola virus infection;

e5) Prepare products that inhibit the proliferation of Ebola virus;

e6) Develop or design or screen products that inhibit the proliferation of Ebola virus;

e7) Preparation of products for the prevention of Ebola virus disease;

e8) Develop or design or screen products for the prevention of Ebola virus disease.

The present invention uses immunoprecipitation and immunofluorescence to confirm the intracellular interaction and co-localization of GP and PAR1. PAR1 activation can promote GP entry into cells, and PAR1 knockdown can inhibit the adsorption of EBOV on the cell surface. In addition, EBOV trVLP infection can recruit PAR1 into viral inclusion bodies and reduce the distribution of PAR1 on the cell surface. Knocking down or knocking out PAR1 significantly inhibited the proliferation of EBOV in cells and the levels of EBOV vRNA in cells and culture supernatants. Overexpression of PAR1 could promote the proliferation of EBOV in cells.

The present invention found that EBOV GP interacts with PAR1 to promote virus entry into cells and PAR1 internalization, and knocking out PAR1 inhibits the adsorption of EBOV trVLP on the cell surface, indicating that PAR1 may participate in the adsorption and entry of EBOV as a receptor or co-receptor. Experiments such as PAR1 knockdown, knockout, restoration and overexpression have confirmed that PAR1 plays an important role in EBOV proliferation, indicating that it can be used as a candidate target for the development of anti-EBOV drugs and has important application value in the treatment of Ebola virus disease.

Description of drawings

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

Figure 2 shows that PAR1 promotes the entry of GP into cells and the adsorption of EBOV on the cell surface. A is the immunofluorescence detection of the effect of PAR1 activation on GP entry into cells. B is the qRT-PCR detection of the effect of PAR1 knockout on EBOV adsorption. C is the effect of PAR1 knockdown on EBOV adsorption. PCR results after removal.

Figure 3 shows that EBOV trVLP infection promotes the internalization of PAR1; where control represents the control untransfected sample, trVLP represents EBOV virus-like particles, and MFI represents the average fluorescence intensity.

Figure 4 shows that EBOV trVLP infection can recruit PAR1 into viral inclusion bodies.

Figure 5 shows that knockdown of PAR1 inhibits the proliferation of EBOV trVLP in cells; A is the qRT-PCR detection of PAR1 knockdown efficiency in HepG2 cells; B is the inhibitory effect of PAR1 knockdown on the proliferation of EBOV trVLP in HepG2 cells; C is immunofluorescence detection The effect of PAR1 knockdown on the proliferation of EBOV trVLP in HepG2 cells; D is the qRT-PCR detection of PAR1 knockdown efficiency in HuH7 cells; E is the inhibitory effect of PAR1 knockdown on the proliferation of EBOV trVLP in HuH7 cells.

Figure 6 shows that knocking down PAR1 inhibits the proliferation of EBOV trVLP in cells and culture supernatants.

Figure 7 shows that overexpression of PAR1 promotes the proliferation of EBOV trVLP in cells.

Detailed ways

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 experimental methods in the following examples, unless otherwise specified, are all conventional methods and are carried out in accordance with the techniques or conditions described in literature in the field or in accordance with product instructions. Materials, reagents, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified. The quantitative experiments in the following examples were repeated three times, and the results were averaged.

The cell lines and plasmids involved in the following examples: HEK293 cells and HuH7 cells were purchased from the Cell Resource Center of the Institute of Basic Medicine, Chinese Academy of Medical Sciences; HepG2 cells were purchased from Yuanjing Biotechnology; plasmids Myc-PAR1 (HG13535-NM) and GP -GFP (VG40304-ACG), Flag-GP (VG40304-NF) were purchased from Sino Biological; Flag-GP1 and Flag-GP2 were purchased from Universal Biosynthesis; pCAGGS-V, pGL3-Promoter were purchased from Youbao Biotechnology; pCAGGS-NP, pCAGGS-VP35, pCAGGS-VP30, pCAGGS-L, pCAGGS-T7, pCAGGS-Tim1 and p4cis-vRNA-Luc in the literature Hoenen T, etal. J. Vis. Exp., 2014). Modeling The Lifecycle Of Ebola Virus Under BiosafetyLevel 2 Conditions With Virus-like Particles Containing TetracistronicMinigenomes is disclosed in Modeling The Lifecycle Of Ebola Virus Under BiosafetyLevel 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes and is available to the public from the Institute of Military Medicine for use in repeating this experiment.

Molecular biology reagents and antibodies involved in the following examples: high-fidelity DNA polymerase KOD FX Neo and SYBR Green Mix were purchased from TOYOBO (FSQ301); cDNA reverse Mix (R333-01) was purchased from Vazyme; The dual-luciferase detection kit was purchased from (E640A) Promega; the transfection reagent Lipofectamine3000 (L3000-015) was purchased from Thermo; the protease inhibitor Cocktail was purchased from Roche (04693132001); DMEM culture medium (C11995500BT), MEM culture Base (C11095500BT) and NEAA (11140-500) were purchased from GIBCO. Fetal bovine serum (FSP500) was purchased from ExCell. HRP-labeled anti-Flag antibody (A8592-1MG) and HRP-labeled anti-Myc antibody (SAB4200742-1VL) were purchased from Sigma; anti-PAR1 (PA5-116040) antibody was purchased from Invitrogen; anti-Flag agarose beads (A2220-1ML) and anti-Myc agarose beads (E6654-1ML) were purchased from Sigma.

The nucleotide sequence corresponding to the PAR1 gene (GeneBank: NM_001992.5, updated on January 29, 2023) in the following examples is shown in Sequence 1, and the amino acid sequence of the PAR1 protein encoded by it is shown in Sequence 2.

Example 1. Immunoprecipitation to detect the interaction between GP and PAR1 in cells.

Taking the co-transfection of Myc-PAR1 and GP-GFP plasmids into 293 cells in a 60 mm dish as an example, transfection was carried out according to the instructions of Lipofectamine 3000 from Thermo Company. The brief is as follows: add 2 μg of Myc-PAR1 and 2 μg of GP-GFP plasmid and 8 μl of P3000. Dilute with 100 μl of opti-MEM; dilute 12 μl of Lipofectamine3000 with 100 μl of opti-MEM, add the diluted plasmid and P3000 mixture drop by drop into the diluted Lipofectamine3000 and mix well; leave it at room temperature for 15 min, and add the plasmid-liposome mixture gradually Add dropwise to cell culture medium.

36-48 hours after transfection, resuspend and wash the cells twice with PBS, centrifuge at 4°C and 1000g/min for 3 minutes to collect the cells; add 400 μl of cell lysis solution (150mM NaCl, 50mM Tris-HCl pH8.0, containing EDTA protease inhibitor 1 slice/50 ml, 1% NP40), lyse on ice for 20 minutes, centrifuge at 1,2000 rpm for 10 minutes at 4°C; transfer the supernatant to a 1.5ml EP tube, add 15 μl of anti-GFP antibody coupled to agarose beads, and rotate at 4°C Incubate for 2 hours for co-immunoprecipitation, centrifuge at 1000g for 3 minutes at 4°C, wash the cells 3 times with 1000 μl of cell lysis solution without protease inhibitors; add an appropriate amount of 1×SDS loading buffer, boil in water bath for 8 minutes, 4°C, 16000g/min Centrifuge for 5 minutes and perform SDS-PAGE electrophoresis and immunoblotting.

Take 10 μl of the sample for SDS-PAGE electrophoresis. The voltage is initially set to 80V. After the Marker bands are clearly separated, adjust the voltage to 120V and continue electrophoresis until bromophenol blue migrates to the bottom of the gel. Stop the electrophoresis; use PVDF membrane with Activate with methanol for 30 seconds, then soak the filter paper in 1x transfer buffer (Tris-HCl 24mM, glycine 5mM, 20% methanol) for 30 minutes; after electrophoresis, place it in half in the order of filter paper - gel - membrane - filter paper from top to bottom. On a dry transfer membrane machine, transfer the membrane at 18V for 2 hours; then block the PVDF membrane at room temperature for 1 hour; wash 3 times with 1×TBST, 5 minutes each time (referred to as washing); add Myc-HRP and GFP-HRP and incubate at room temperature for 1 hour; wash with TBST. ECL development.

The results are shown in Figure 1, A. The immunoprecipitation experiment confirmed the interaction between EBOV GP and PAR1 in cells.

Example 2: Immunofluorescence detection of co-localization of GP and PAR1 in cells.

HepG2 cells were transfected with 1 μg Myc-PAR1 and 1 μg GP-GFP or GFP using Lipofectamine 3000 from Thermo Company (the operation method is shown in Example 1). After 48 hours of transfection, wash with PBS three times to absorb the remaining liquid, add 4% paraformaldehyde and fix at 37°C for 30 minutes; after washing with PBS, add 0.3% Triton X-100 (prepared with 1×PBS) to make holes for 15 minutes; add 2 ml of blocking solution (1×PBS containing 5% goat serum) blocked for 30 min at 37°C; after washing with PBS, add anti-PAR1 antibody (1:50 dilution of blocking solution), and incubate at 4°C overnight or at room temperature for 1 hour; wash cells 3 times with 1×PBST, 10 min each time (referred to as PBST wash); add TRITC-labeled goat anti-rabbit antibody (1:00 dilution in blocking solution) to the cells, and incubate for 1 hour at room temperature in the dark; after washing with PBST, add 10 μl of mounting solution containing DAPI (1 μg/ml) , after standing for 15 minutes, the distribution of PAR1 and GFP in the cells was detected by laser confocal microscopy (Carl ZeissLSM800).

The results are shown in B in Figure 1. The study found that GP and PAR1 co-localize on the cell membrane surface through immunofluorescence technology.

Example 3. Immunofluorescence detection of the effect of PAR1 activation on GP entry into cells.

HepG2 cells were transfected with 1 μg of GP-GFP or GFP using Lipofectamine 3000 from Thermo Company (the operation method is as shown in Example 1). 48 hours after transfection, add 50 μmol/L PAR1 agonist peptide (AP, TFLLRN) for 10 minutes, then wash and fix the cells, perforate, block, incubate PAR1 antibody and TRITC fluorescent secondary antibody (the operation method is as shown in Example 2), and detect Effects of PAR1 activation on GFP distribution in cells.

The results are shown in A in Figure 2. The results show that after PAR1 is activated by agonists and internalized into cells, GP located on the cell membrane surface also enters the cells and co-localizes with the internalized PAR1.

Example 4. qRT-PCR detection of the effect of PAR1 knockout on the adsorption of EBOV trVLP on the cell surface.

In order to verify the effect of PAR1 on the adsorption of EBOV trVLP on the cell surface, this example used CRISPR/Cas9 technology to construct a HepG2 cell line in which 299 bp of the PAR1 gene was knocked out (the knockout sequence is shown in positions 53-351 of Sequence 9 in the sequence listing). , the brief process is as follows: (1) sgRNA construction: Use the sgRNA sequence online design website (http://crispr.mit.edu/) to design two pairs of sgRNA targeting PAR1 (sgRNA1:AAATGACCGGGGATCTAAGGTGG, as shown in sequence 7 in the sequence listing ; sgRNA2: TGCAGCATGTACACCACCGCCGG, as shown in sequence 8 in the sequence listing), add the sticky end CACCG to the 5' end of the sense strand of sgRNA, and add the sticky end AAAC to the 5' end of the antisense strand; (2) The synthesized sgRNA sequence (Beijing No. Spe Genome Research Center Co., Ltd.) was annealed and cloned into the pSpCas9 (BB)-2A-Puro (PX459) vector (for the method, refer to the Molecular Cloning Experiment (Third Edition) Guide). (3) Co-transfect the two pairs of recombinant plasmids into HepG2 cells, and select single clones with 1 μg/ml puromycin 48 hours after transfection; (4) Limit the obtained clones to a 96-well plate, and expand and culture to 24-well plate, 6-well plate; (5) Screening of positive clone cell lines: Design upstream and downstream primers for the sgRNA target sequence, amplify the upstream and downstream fragments containing the target gene fragment; agarose gel electrophoresis detection and sequencing confirmation, screening PAR1 knockout cell line.

Design primer F1 upstream of sgRNA1 according to the nucleic acid sequence: GGGTAGATCTCTGAAAACCTATC, as shown in sequence 10 in the sequence listing; design primer R1 downstream of sgRNA2: GATGTTGAGCCCGGGCACC, as shown in sequence 11 in the sequence listing; design primer R2 between gRNA1 and gRNA2: CTGACTAAAACACTCCGGTG, as shown Sequence 12 is shown in the sequence list; two pairs of primers F1/R1 and F1/R2 were designed for PCR amplification to verify whether the knockout was successful. The PCR results are shown in C in Figure 2. The F1/R1 primer pair was used to amplify the wild type. The nucleic acid electrophoresis size of cells (WT) was 933 bp (lane 1), the nucleic acid electrophoresis size of amplified mutant cells (#D12) was 634 bp (lane 2), and the nucleic acid electrophoresis size of wild-type cells amplified using the F1/R2 primer pair was The size was 527bp (lane 3), and the nucleic acid electrophoresis size of the amplified mutant cells was 0bp (lane 4); the results showed that the PAR1 gene was successfully knocked out.

EBOV minimal genome system related plasmids were transfected into HepG2 cells respectively: pCAGGS-NP (125ng), pCAGGS-VP35 (125ng), pCAGGS-VP30 (75ng), pCAGGS-L (1000ng), p4cis-vRNA-Rluc (250ng) and pCAGGS-T7 (250ng) (the operation method is shown in Example 1). The cell supernatant was collected 48 hours after transfection to obtain Ebola virus-like particles (trVLP). EBOV trVLP supernatant was added to HepG2 cells and PAR1 knockout cells and adsorbed at 4°C for 1 hour, and then pre-cooled PBS was used to wash the EBOV trVLP that was not adsorbed in the cells. Collect cells and extract viral genomic RNA in a biosafety cabinet according to QIAamp Viral RNAMini Kit (Qiagen) instructions (52906). Subsequently, the cDNA sequence was synthesized by reverse transcription according to the Novozantreverse Transcription Kit (R333-01) (totol RNA 1μg, 5×All-in-one qRT-PCRSuperMix 4μl, Enzyme Mix 1μl, ddH ₂ O to 20μl; program 50℃ , 15min; 85℃, 5s). Through QuantStudio6 fluorescence quantitative PCR, the SYBR Green Mix (Toyobo) method was used to detect the RNA transcription level of VP40 in cells (VP40F: GGAGGCCATATACCCTGTCAGGTC, as shown in sequence 13 in the sequence listing; VP40R: GCCTGGTGTGTGGCTGGCAT, as shown in sequence 14 in the sequence listing; GAPDH -F: AAGGTCATCCCTGAGCTGAAC, as shown in sequence 15 in the sequence listing; GAPDH-R: ACGCCTGCTTCACCACCTTCT, as shown in sequence 16 in the sequence listing).

The results are shown in Figure 2, B. The results show that PAR1 knockout can significantly inhibit the adsorption ability of EBOV on the cell surface.

Example 5. Flow cytometry to detect the effect of EBOV trVLP infection on the distribution of PAR1 on the cell surface.

HepG2 cells were transfected with Myc-PAR1 500ng and EBOV minimal genome system related plasmids: pCAGGS-NP (125ng), pCAGGS-VP35 (125ng), pCAGGS-VP30 (75ng), pCAGGS-L (1000ng), p4cis-vRNA- Rluc (250ng) and pCAGGS-T7 (250ng) (the operation method is shown in Example 1). Collect cells 48 hours after transfection, wash twice with 1×PBS, add Myc antibody (1:50 dilution) and incubate for 1 hour, then incubate with FITC fluorescent secondary antibody, and statistically analyze the effect of EBOV trVLP infection on PAR1 by flow cytometry (MilliporeImageStreamX MarkII) Effect of membrane surface distribution.

The results are shown in Figure 3. Through flow cytometry, it was found that EBOV trVLP infection can significantly reduce the distribution of PAR1 on the cell membrane surface in HepG2.

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

EBOV minimal genome system related plasmids were transfected into HepG2 cells: pCAGGS-NP (125ng), pCAGGS-VP35 (125ng), pCAGGS-VP30 (75ng), pCAGGS-L (1000ng), p4cis-vRNA-Rluc (250ng) and pCAGGS-T7 (250ng) (the operation method is shown in Example 1). Cells were collected at 0h, 6h, 12h, 18h, 24h, 36h and 48h after transfection. The cells were then washed and fixed, perforated, blocked, and incubated with PAR1 and VP35 antibodies, as well as FITC and TRITC fluorescent secondary antibodies (the operation method is shown in Example 2), and the effects of EBOV infection at different times on the distribution of PAR1 in cells were detected.

The results are shown in Figure 4. HepG2 cells were infected with EBOV trVLP at different time points (0h, 6h, 12h, 18h, 24h, 36h and 48h) to detect the dynamic changes in the distribution of PAR1 in the cells. The results showed that 18 hours after EBOV infection, PAR1 was gradually recruited into viral inclusion bodies, the site of viral replication.

Example 7. Effect of PAR1 knockdown on the proliferation of EBOV trVLP in cells.

EBOV minimal genome system: This study utilizes the EBOV minimal genome system that can be operated in a biosafety level two laboratory to detect virus levels in cells through luciferase activity. The experimental operation process is briefly as follows: on day 1, inoculate virus production cells (referred to as p0) in a ⁶ -well plate at 3x10 cells per well, and place them in an incubator at 37°C and 5% _CO2 for static culture; on day 2 On the same day, plasmids pCAGGS-NP (125ng), pCAGGS-VP35 (125ng), pCAGGS-VP30 (75ng), pCAGGS-L (1000ng), p4cis-vRNA-Rluc (250ng) and pCAGGS-T7 (250ng) were mixed according to the instructions of lipofectamine3000 Transfected into p0 cells; on the 3rd day, the p0 supernatant was replaced with fresh medium containing 5% FBS. On the 4th day, the virus-targeted cells (referred to as p1) were inoculated into a ⁶ -well plate at 3x105 per well; on the 5th day, the plasmids pCAGGS-NP (125ng), pCAGGS-VP35 (125ng), pCAGGS-VP30 ( 75ng), pCAGGS-L (1000ng) and pCAGGS-Tim1 (250ng) were transfected into p1 cells according to lipofectamine3000 instructions; on day 6, the p0 cell supernatant was collected, and a dual-luciferase detection kit was used to detect EBO in p0 cells. Replication of the viral minimal genome. The supernatant of p1 cells was replaced with the supernatant of p0 cells; on the 7th day, the supernatant of p1 was replaced with 5% FBS culture medium, and p1 was harvested after continuing to culture for 72 hours. If you need to continue to pass the virus to obtain p2, the method is the same as the p1 acquisition process.

Luciferase activity detection: Use Thermo Lipofectamine 3000 to transfect PAR1 siRNA in HepG2 or HuH7 cells (sense: 5'- GGCUACUAUGCCUACUACUTT-3', as shown by replacing T with U in sequence 3; antisense: AGUAGUAGGCAUAGUAGCCUU-3', As shown in sequence 4, T is replaced by U) or Scr siRNA (sence, 5'-UUCUCCGAACGUGUCACGUTT-3', as shown in sequence 5, T is replaced by U; antisense:5'-ACGUGACACGUUCGGAGAATT-3', as shown in sequence 6 T is replaced by U (shown as U), and 6 hours after transfection, the EBOV minimal genome system-related plasmid and firefly reporter gene pGL3-Basic (25ng) were transfected. Collect p0 cells in the minimal genome system. After washing with PBS, add 500 μl PLB lysis buffer and lyse on a shaking table for 15 min. After centrifugation, take 20 μl of cell lysis supernatant and add it to 100 μl Luciferase Assay Reagent Ⅱ. After mixing, add TD-20/20 fluorescence Measure the luminescence value RLU1 with a photometer, then add 100 μl Stop&Glo Reagent to measure the fluorescence luminescence value RLU2. The relative luciferase activity value of RLU2/RLU1 evaluated the virus level, thereby confirming the effect of PAR1 knockdown on the proliferation of EBOV trVLP in cells.

Immunofluorescence detection: Use Thermo's Lipofectamine 3000 to transfect PAR1 siRNA or Scr siRNA into HepG2 or HuH7 cells, and then transfect EBOV minimal genome system-related plasmid 6 hours after transfection. Collect the cells 48 hours after transfection, and then wash and fix the cells. Puncture, block, and incubate PAR1 and VP35 antibodies, as well as FITC and TRITC fluorescent secondary antibodies (the operation method is shown in Example 2), and analyze the effect of PAR1 knockdown on the proliferation of EBOV trVLP through the expression of VP35.

The results are shown in Figure 5. This example uses the EBOV minimal genome system to detect the effects of PAR1 knockdown and knockout on the proliferation of EBOV trVLP through luciferase activity experiments. PAR1 siRNA was transfected into HepG2 cells to knock down endogenous PAR1 in HepG2 cells (as shown in Figure 5, A), and then transfected with EBOV minimal genome-related plasmid. The luciferase activity experiment found that knocking down PAR1 significantly inhibited the expression of PAR1 in HepG2 cells. EBOV trVLP proliferated (~2.7-fold) (shown in Figure 5, B). Immunofluorescence experiments can intuitively show that PAR1 knockdown significantly inhibits the proliferation of EBOV trVLP in cells (indicated by VP35) (shown in Figure 5, C). Secondly, knocking down PAR1 in HuH7 cells also significantly inhibited EBOV trVLP proliferation (~3.0-fold) (shown in Figure 5, D and Figure 5, E).

Example 8. Effect of PAR1 knockout on the proliferation of EBOV trVLP in cells.

Luciferase activity detection: HepG2 and PAR1 knockout (PAR1 KO) cells were transfected with EBOV minimal genome-related plasmid and firefly reporter gene pGL3-Basic (25ng). The dual-luciferase activity detection method is the same as above (consistent with the luciferase activity detection method in Example 7), and the effect of PAR1 knockout on the proliferation of EBOV trVLP in cells is analyzed.

qRT-PCR detection: HepG2 and PAR1 knockout (PAR1 KO) cells were transfected with EBOV minimal genome-related plasmid, cells and culture supernatants were collected 120 hours after transfection, RNA was extracted, and qRT-PCR detection was performed after inversion (the method is the same as The method is the same as in Example 4), and the effect of PAR1 knockdown on EBOV RNA in cells and cell supernatants was analyzed.

The results are shown in Figure 6. This example examined the effect of PAR1 knockout on the proliferation of EBOV trVLP. HepG2 and PAR1 knockout cells were transfected with EBOV minimal genome-related plasmids. The luciferase results showed that: they were comparable to HepG2 wild-type cells. Compared with PAR1 knockout, EBOV trVLP proliferation decreased approximately 2-fold and 12-fold in p0 and p1 generation cells (shown in Figure 6, A). When Myc-PAR1 and EBOV minimal genome-related plasmids were transfected in PAR1 knockout cells, the results showed that restoring PAR1 expression in PAR1 knockout cells could significantly promote the proliferation of EBOV trVLP (shown in Figure 6, B). In addition, qRT-PCR experiments revealed that PAR1 knockdown could significantly suppress EBOV RNA levels in cells and culture supernatants (∼12-fold and ∼16-fold) (shown in Figure 6, C and Figure 6, D).

Example 9. Effect of PAR1 overexpression on the proliferation of EBOV trVLP in cells.

Luciferase activity detection: Myc-PAR1 (or Myc) was transfected into HepG2 cells, and 6 hours after transfection, the EBOV minimal genome system-related plasmid and the firefly reporter gene pGL3-Basic (25ng) were transfected (method and Example 7) The method is consistent), and the effect of PAR1 overexpression on the proliferation of EBOV trVLP in cells was analyzed.

The results are shown in Figure 7. Overexpression of PAR1 can promote the proliferation of EBOV trVLP in cells (∼3.0-fold).

Combining the results of the above examples, the present invention found that the interaction between EBOV GP and PAR1 promotes virus entry into cells and PAR1 internalization, and knocking out PAR1 inhibits the adsorption of EBOV trVLP on the cell surface, indicating that PAR1 may participate in EBOV as a receptor or coreceptor. Adsorption and entry. Experiments such as PAR1 knockdown, knockout, restoration and overexpression have confirmed that PAR1 plays an important role in EBOV proliferation, indicating that it can be used as a candidate target for the development of anti-EBOV drugs and has important application value in the treatment of Ebola virus disease.

The present invention has been described in detail above. For those skilled in the art, the present invention can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the invention and without performing unnecessary experiments. Although specific embodiments of the present invention have been shown, it should be understood that further modifications can be made to the invention. In short, based on the principles of the present invention, this application is intended to include any changes, uses, or improvements to the present invention, including changes that depart from the scope disclosed in this application and are made using conventional techniques known in the art. Some essential features may be applied within the scope of the appended claims below.

Claims (2)

1. Application of substances that knock out PAR1 gene or substances that inhibit PAR1 gene expression in any one of the following a1)-a4), wherein the nucleotide sequence of PAR1 gene is as shown in Sequence 1, The substance that inhibits PAR1 gene expression is siRNA formed by the annealing of two single strands with the sequences GGCUACUAUGCCUACUACUTT and AGUAGUAGGCAUAGUAGCCUU; The material that knocks out the PAR1 gene is the CRISPR/Cas9 gene editing system that knocks out the PAR1 gene; The sgRNA target sequence in the CRISPR/Cas9 gene editing system for knocking out the PAR1 gene is sequence 7 or sequence 8: a1) Prepare products for the treatment or auxiliary treatment of Ebola virus disease; a2) Prepare products that inhibit Ebola virus infection; a3) Prepare products that inhibit the proliferation of Ebola virus; a4) Preparation of products for the prevention of Ebola virus disease. 2. The application of substances that inhibit PAR1 protein activity or substances that reduce PAR1 protein content in any of the following b1)-b4), wherein the amino acid sequence of PAR1 protein is shown in sequence 2, and the substance that inhibits PAR1 protein activity The substance or substance that reduces the PAR1 protein content is siRNA formed by the annealing of two single strands with the sequences GGCUACUAUGCCUACUACUTT and AGUAGUAGGCAUAGUAGCCUU: b1) Preparation of products for the treatment or auxiliary treatment of Ebola virus disease; b2) Prepare products that inhibit Ebola virus infection; b3) Prepare products that inhibit the proliferation of Ebola virus; b4) Preparation of products for the prevention of Ebola virus disease.