Application of biomacromolecular protein CXCL4 in preparation of anti-new coronavirus drugs

Abstract

The invention discloses an application of a biomacromolecular protein CXCL4 in preparation of an anti-new coronavirus drug, the biomacromolecular protein CXCL4 capable of inhibiting replication of the new coronavirus at a cellular level is obtained through screening, and the biomacromolecular protein CXCL4 comprises a secretion-deficient biomacromolecular protein delta sp-CXCL4; the biomacromolecular protein can inhibit four SARS-CoV-2 subtype pseudoviruses from entering cells, inhibit the replication of SARS-CoV-2 and inhibit the infection of SARS-CoV-2 at the stage that the viruses enter the cells, thereby laying a theoretical foundation and providing a new thought for mining novel anti-new coronavirus drugs.

Description

(1) Technical field

[0001] The present invention belongs to the field of biomedicine and relates to the application of a biological macromolecular protein CXCL4 in the preparation of anti-new coronavirus drugs. (2) Background technology

[0002] The outbreak of SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) in late 2019 caused a global pandemic. SARS-CoV-2 is a single-stranded, positive-sense RNA with a genome length of approximately 30 kilobases (KB) consisting of four structural proteins (nucleoprotein N, membrane protein M, spike protein S, and envelope protein E) and 16 nonstructural proteins (nsp1-16). This high variability of the novel coronavirus is a technical challenge in developing broad-spectrum anti-SARS-CoV-2 drugs.

[0003] Natural antiviral factors are an important source of antiviral drugs. Natural antiviral factors have low toxicity and no obvious side effects on the human body. Therefore, it is necessary to find natural biomolecules that can inhibit the replication of the new coronavirus at the cellular level. (3) Summary of the invention

[0004] The purpose of the present invention is to provide an application of a biomacromolecule protein CXCL4 in the preparation of anti-new coronavirus drugs. The biomacromolecule protein CXCL4 can inhibit the replication of the new coronavirus at the cellular level, providing new ideas for the research of anti-new coronavirus drugs.

[0005] The technical solution adopted in the present invention is:

[0006] The present invention provides an application of a biomacromolecule protein CXCL4 in the preparation of anti-new coronavirus drugs.

[0007] Furthermore, the drug includes a secretion-defective biomacromolecule protein δsp-CXCL4, which is a CXCL4 truncation lacking a signal peptide region, and the δsp-CXCL4 monomer is obtained by expression and purification in Escherichia coli.

[0008] Furthermore, the amino acid sequence of the biomacromolecule protein CXCL4 is shown in SEQ ID NO.1.

[0009] Furthermore, the amino acid sequence of the secretion-deficient biomacromolecule protein δsp-CXCL4 is shown in SEQ ID NO.2.

[0010] Furthermore, the drug also includes a medically acceptable carrier.

[0011] Furthermore, the pharmaceutical dosage form is one or more of tablets, capsules, granules, dispersions, injections or sprays.

[0012] Furthermore, the application includes preparing one or more of an antiviral product, a virus-inhibiting product, or a product for preventing viral infection; the virus-inhibiting product is a product that inhibits viral replication. The content of the biomacromolecule protein CXCL4 or the secretion-deficient biomacromolecule protein δsp-CXCL4 in the product is not less than 0.5 nM.

[0013] Furthermore, the virus includes SARS-CoV-2 strains, not limited to strain 19nCoVCDC-Tan-HB01 (HB01) or strain SARS-CoV-2-BF7.14.

[0014] Compared with the existing technology, the beneficial effects of the present invention are mainly reflected in: the present invention screened and obtained a biomacromolecule protein CXCL4 that can inhibit the replication of the new coronavirus at the cellular level, including the secretion-defective biomacromolecule protein δsp-CXCL4; the biomacromolecule protein of the present invention can inhibit the entry of four SARS-CoV-2 subtypes of pseudoviruses into cells, inhibit the replication of SARS-CoV-2, and inhibit the infection of SARS-CoV-2 at the stage when the virus enters the cell, laying a theoretical foundation for the exploration of new anti-new coronavirus drugs and providing new ideas. (IV) Description of the accompanying drawings

[0015] Figure 1 , the effect of CXCL4 on cells infected with four SARS-CoV-2-S subtype pseudoviruses; (A) fluorescence microscopy, (B) flow cytometry.

[0016] Figure 2 , Effects of δsp-CXCL4 on cells infected with four SARS-CoV-2-S subtype pseudoviruses. (A) Fluorescence microscopy, (B) flow cytometry.

[0017] Figure 3 , immunoblotting images of different concentrations of CXCL4 protein inhibiting different SARS-CoV-2 strains in vitro.

[0018] Figure 4 , immunoblotting images of different concentrations of δsp-CXCL4 protein inhibiting different SARS-CoV-2 strains in vitro.

[0019] Figure 5 , immunoblot images showing that CXCL4 plays an inhibitory role in the process of SARS-CoV-2 invading cells, A represents 19nCoVCDC-Tan-HB01 (HB01), and B represents SARS-CoV-2-BF7.14.

[0020] Figure 6 , immunoblotting of CXCL4-Flag and SARS-CoV-2-E-HA. (V) Specific implementation methods

[0021] The present invention is further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto:

[0022] The amino acid sequence of CXCL4 is shown in SEQ ID NO. 1:

[0023] SSAAGFCASRPGLLFLGLLLLPLVVAFASAEAEEDGDLQCLCVKTTSQVRPRHITSL EVIKAGPHCPTAQLIATLKNGRKICLDLQAPLYKKIIKKLLES*

[0024] The amino acid sequence of δsp-CXCL4 is SEQ ID NO. 2:

[0025] MEAEEDGDLQCLCVKTTSQVRPRHITSLEVIKAGPHCPTAQLIATLKNGRKICLDLQAPLYKKIIKKLLESYPYDVPDYA*

[0026] Example 1: CXCL4 inhibits SARS-CoV-2-S pseudovirus infection in Vero-E6 cells

[0027] (1) Construction of pseudoviral plasmids of different subtypes of SARS-CoV-2-S:

[0028] The preparation method for the SARS-CoV-2-S pseudoviruses of four subtypes (BA.4 / 5, BF.7, XBB.1.5, and EG.5) followed the method described by Academician Gao Fu (Li et al., 2023). First, the S protein sequence was constructed into the pCAGGS vector, the C-terminal 18 amino acids of each S protein were truncated, and codon optimization was performed to ensure expression in HEK-293T cells. Four pseudovirus vectors were obtained: pCAGGS-SARS-CoV-2-S-BA.4 / 5, pCAGGS-SARS-CoV-2-S-BF.7, pCAGGS-SARS-CoV-2-S-XBB.1.5, and pCAGGS-SARS-CoV-2-S-EG.5.

[0029] (2) Preparation of different subtypes of SARS-CoV-2-S pseudoviruses

[0030] 5×10 6HEK-293T cells were plated in a 10 cm cell culture dish in a 10 mL DMEM culture medium containing 10% fetal bovine serum. After the cells adhered to the wall, they were transfected with the four pseudovirus vectors pCAGGS-SARS-CoV-2-S BA.4 / 5, BF.7, XBB.1.5, and EG.5 constructed in step (1). After 24 hours, VSV-ΔG-GFP virus was added to infect the cells. After 2 hours of infection, the cells were inoculated with anti-VSV-G antibody (I1HybridomaATCC- The culture medium was replaced with fresh DMEM supplemented with 10% fetal bovine serum (FBS). After incubation at 37°C for 30 hours, the supernatant was collected and sterilized using a 0.22 μm filter (Millipore, Cat# SLHP033RB). The filtrate was aliquoted and stored frozen at -80°C to obtain the supernatants of the four SARS-CoV-2-S pseudovirus subtypes.

[0031] (3) Construction and transfection of CXCL4 plasmid:

[0032] Construction of CXCL4-HA plasmid: The CXCL4 gene sequence with the HA tag was inserted into the VR1012 vector sequence to construct the CXCL4 protein expression plasmid VR1012-CXCL4-HA.

[0033] HEK-293T cells were transfected with VR1012-CXCL4-HA plasmid. After 48 hours, the supernatant was collected, concentrated, and the concentration of CXCL4 was detected using an ELISA kit to obtain a CXCL4 protein solution.

[0034] (4) CXCL4 inhibits SARS-CoV-2-S pseudovirus infection

[0035] Vero-E6 was added at 1×10 4 Cells were plated into 12-well plates (1 mL of DMEM culture medium containing 10% fetal bovine serum was added to each well) until the cells were completely attached. The CXCL4 protein solution prepared in step (3) was added to the final concentrations of 0 nM, 0.5 nM, and 1 nM, respectively, and incubated at 37°C for 4 hours. Each concentration of CXCL4 was added to the supernatant of the four subtypes of pseudovirus prepared in step (2) (pseudovirus SARS-CoV-2-S BA.4 / 5, BF.7, XBB.1.5, EG.5 subtypes) at an MOI of 0.01, 0.01, 0.05, and 0.05. After 24 hours of infection, the infection rate of the pseudovirus SARS-CoV-2-S was observed by fluorescence microscopy. The results are shown in the figure. Figure 1 In Figure A, CXCL4 was found to inhibit the infection of cells with pseudoviruses of all four SARS-CoV-2 subtypes. The ratio of GFP-luminescent cells to the total number of cells was detected by flow cytometry. Figure 1In Figure B, it was found that as the concentration of CXCL4 increased, the percentage of cells infected with the pseudoviruses of the four SARS-CoV-2 subtypes gradually decreased, indicating that CXCL4 can inhibit the entry of the pseudoviruses of the four SARS-CoV-2 subtypes into cells.

[0036] Example 2: In Vero-E6 cells, δsp-CXCL4 inhibits the entry of SARS-CoV-2-S pseudovirus into cells

[0037] Secretion-deficient protein δsp-CXCL4: δsp-CXCL4 with a his tag was cloned into the prokaryotic expression vector pet-28a, transformed into E coli.BL21 competent cells, single clones were selected and cultured, and expression was induced for 16 hours under 0.5 mM isopropylthiogalactoside (IPTG) at 16°C. The precipitate was centrifuged at 6000g for 1 hour, and the precipitate was collected and disrupted using an ultrasonic disruptor (200W ultrasonic for 30 minutes, with 3 seconds of disruption and 3 seconds of interval). The fragmentation mixture was purified by Ni-column affinity chromatography and eluted with 100 mM, 150 mM and 200 mM imidazole-containing eluents (20 mM Tris, 200 mM NaCl, pH = 8) at a rate of 2 mL / min for 10 column volumes. The his-tagged δsp-CXCL4 protein was collected and concentrated. The concentrated protein was analyzed by Human-CXCL4 ELISA. Kit was used to detect its concentration.

[0038] Vero-E6 was added at 1×10 4 1 / well were plated in a 12-well plate. After the cells were completely attached, δsp-CXCL4 protein was added to a final concentration of 0 nM, 10 nM, and 30 nM, respectively. The cells were incubated at 37 ° C for 4 hours, and the four subtypes of pseudovirus supernatants (SARS-CoV-2-S BA.4 / 5, BF.7, XBB.1.5, EG.5 subtypes) prepared by the method of Example 1 were infected with MOI of 0.01, 0.01, 0.05, and 0.05, respectively. After 24 hours of infection, the infection rate of the pseudovirus SARS-CoV-2-S was observed by fluorescence microscopy. It was found that with the increase of δsp-CXCL4 concentration, the infection rate of the four subtypes of SARS-CoV-2-S gradually decreased, indicating that δsp-CXCL4 can inhibit the pseudovirus infection of cells by the four SARS-CoV-2 subtypes ( Figure 2 Middle A). By detecting the proportion of GFP-luminescent cells to the total number of cells using flow cytometry, it was found that as the concentration of δsp-CXCL4 increased, the percentage of cells infected with the four SARS-CoV-2 subtypes of pseudovirus gradually decreased, indicating that δsp-CXCL4 can inhibit the entry of the four SARS-CoV-2 subtypes of pseudovirus into cells ( Figure 2 Middle B).

[0039] Example 3: CXCL4 inhibits SARS-CoV-2 replication in Vero-E6 cells

[0040] Vero-E6 was used as a 5×10 5 Cells were plated into 6-well plates (the volume of DMEM medium with 10% fetal bovine serum per well was 2 mL), and CXCL4 protein solutions at concentrations of 0 nM, 0.5 nM, 1.0 nM, 2.0 nM, and 4.0 nM prepared by the method in Example 1 were added 12 hours later, and incubated at 37 ° C for 4 hours. After incubation, the cells were moved into a BSL-3 laboratory and infected with SARS-CoV-2 strains: 19nCoVCDC-Tan-HB01 (HB01) or SARS-CoV-2-BF7.14 at an MOI of 0.01. After 24 hours of infection, the proteins in the cell samples were subjected to immunoblotting experiments. Western blot experiments were performed using antibodies: anti-SARS-CoV-2-N antibody (Genetex, GTX635679), anti-SARS-CoV-2-M antibody (Genetex, GTX636246), anti-SARS-CoV-2-E antibody (Proteintech, 28904-1-AP), tubulin antibody (Proteintech, 10068-1-AP), horseradish peroxidase-conjugated mouse secondary antibody (Jackson, 115-035-062) and rabbit secondary antibody (Jackson, 111-035-045), and ECL color development substrate. The results showed that CXCL4 could inhibit the replication of SARS-CoV-2. As the concentration of CXCL4 increased, the expression of SARS-CoV-2-N, M, and E proteins gradually decreased ( Figure 3 ).

[0041] Example 4: δsp-CXCL4 inhibits SARS-CoV-2 replication in Vero-E6 cells

[0042] Vero-E6 was used as a 5×10 5Cells were plated in 6-well plates (the volume of DMEM culture medium with 10% fetal bovine serum in each well was 2 mL). After 12 hours, 0 nM, 10 nM, 20 nM, and 30 nM δsp-CXCL4 protein solutions prepared by the method of Example 2 were added, and incubated at 37 ° C for 4 hours. After incubation, the cells were moved into the BSL-3 laboratory and infected with SARS-CoV-2 strains: 19nCoVCDC-Tan-HB01 (HB01) or SARS-CoV-2-BF7.14 at an MOI of 0.01. After 24 hours of infection, the SARS-CoV-2-N, M, and E proteins in the cell samples were immunoblotted. The results showed that δsp-CXCL4 can inhibit the replication of SARS-CoV-2. As the concentration of δsp-CXCL4 increases, the expression of SARS-CoV-2-N, M, and E proteins gradually decreases ( Figure 4 ).

[0043] Example 5. CXCL4 plays a role in inhibiting the entry of SARS-CoV-2 into cells.

[0044] Untreated: Vero-E6 cells (1×10 5 Cells were infected with SARS-CoV-2 strains: 19nCoVCDC-Tan-HB01 (HB01) or SARS-CoV-2-BF7.14 at an MOI of 0.01, and the virus was washed off with PBS after 2 hours and cultured for 24 hours.

[0045] Pre-treatment: The CXCL4 protein solution (1 nM) prepared by the method of Example 1 was incubated with Vero-E6 cells (1×10 5 The cells were incubated for 2 hours, and then infected with SARS-CoV-2 strains: 19nCoVCDC-Tan-HB01 (HB01) or SARS-CoV-2-BF7.14 at an MOI of 0.01. After 2 hours, the virus was washed away with PBS and cultured for 24 hours.

[0046] Incubation treatment: The CXCL4 protein solution (1 nM) prepared by the method of Example 1 was mixed with SARS-CoV-2 strains: 19nCoVCDC-Tan-HB01 (HB01) or SARS-CoV-2-BF7.14, and then infected Vero-E6 cells (1×10 5 The virus was washed off with PBS after 2 hours and cultured for 24 hours.

[0047] Post-entry treatment: Vero-E6 cells (1×10 5 / well, 12-well plate) was first infected with SARS-CoV-2 strain: 19nCoVCDC-Tan-HB01 (HB01) or SARS-CoV-2-BF7.14 at an MOI of 0.01. After 2 hours, the virus was washed off with PBS. After 4 hours, the CXCL4 protein solution (1 nM) prepared by the method in Example 1 was added and incubated. After 2 hours, the CXCL4 protein was washed off with PBS and cultured for 24 hours.

[0048] The cells were harvested and immunoblotting was performed. Anti-SARS-CoV-2-N / M / E antibodies and tubulin antibodies, horseradish peroxidase-conjugated mouse secondary antibodies and rabbit secondary antibodies were used. Figure 5 As shown: Compared with the untreated group, the virus yield in the pretreatment group was significantly reduced, the virus yield in the co-incubation group also decreased, and the virus yield in the post-treatment group did not decrease significantly. The results show that CXCL4 inhibits SARS-CoV-2 infection at the stage of viral entry into cells.

[0049] Example 6: Immunoprecipitation experiments revealed that CXCL4 binds to SARS-CoV-2-E protein.

[0050] HEK-293T cells were cultured at 5 × 10 5 Cells were plated into 6-well plates (2 mL of DMEM medium containing 10% fetal bovine serum per well). Figure 6 As shown, 1.5 μg of CXCL4-Flag (flag tag DYKDDDK added to the C-terminus of CXCL4 protein) and SARS-CoV-2-E-HA (HA tag YPYDVPDYA added to the C-terminus of SARS-CoV-2-E protein) were transfected respectively. After 48 hours, the cell samples were collected, the cell culture medium was washed with PBS, the cell culture plate was placed on ice, and the cells were lysed with 1 mL / well of lysis buffer containing protease inhibitors (50 mM Tris, 150 mM NaCl, 0.5% NP-40). The cells were flipped continuously in a 4°C cold room and lysed for more than 2 hours. After the lysis was completed, the sample was taken out and placed in a 4°C centrifuge at 12000g for 10 minutes, and the supernatant was collected.

[0051] Input sample: Take 60 μL of the collected supernatant, add 20 μL of 4× loading buffer, mix well and use as the input sample. After vortexing and mixing, boil at 100°C for 30 min and store at -20°C until use.

[0052] IP sample preparation: 800 μL of the remaining supernatant was added to 3 μL of either Flag antibody (Sigma, F1804) or HA antibody (Biolegend, 901514). The mixture was incubated on a shaker at 4°C for 30 minutes. The sample was then added to 30 μL of Protein G agarose beads (Roche, 11243233001) and incubated at 4°C for 4 hours. After incubation, the beads were centrifuged at 800 g for 1 minute. The pellet was collected and washed with 1 mL of pre-cooled eluent (20 mM Tris, 100 mM NaCl, 0.1 mM EDTA). Repeat this five times. The eluate was then mixed with 4× Loading Buffer and boiled for 30 minutes.

[0053] The input and IP samples were analyzed by immunoblotting. The results showed that CXCL4-Flag could capture SARS-CoV-2-E-HA (A), and conversely, SARS-CoV-2-E-HA could also capture CXCL4-Flag (B). This suggests that CXCL4 binds to SARS-CoV-2-E, and that the mechanism by which CXCL4 inhibits SARS-CoV-2 replication may be by binding to SARS-CoV-2-E and blocking viral entry into cells.

Claims

1. Application of a biological macromolecular protein CXCL4 in the preparation of anti-new coronavirus drugs.

2. The use according to claim 1, characterized in that The amino acid sequence of the biomacromolecule protein CXCL4 is shown in SEQ ID NO.

1.

3. The use according to claim 1, characterized in that The drug comprises a secretion-deficient biomacromolecule protein δsp-CXCL4, wherein the secretion-deficient biomacromolecule protein δsp-CXCL4 is a truncated form of CXCL4 lacking a signal peptide region.

4. The use according to claim 3, characterized in that The amino acid sequence of the secretion-deficient biomacromolecule protein δsp-CXCL4 is shown in SEQ ID NO.

2.

5. The use according to any one of claims 1 to 4, characterized in that: The medicament further includes a medically acceptable carrier.

6. The use according to any one of claims 1 to 4, characterized in that: The pharmaceutical dosage form is one or more of tablets, capsules, granules, dispersions, injections or sprays.

7. The use according to any one of claims 1 to 4, characterized in that: The application includes one or more of preparing antiviral products, preparing virus-inhibiting products or preparing products for preventing viral infection; the virus-inhibiting products are products that inhibit viral replication.

8. The use according to claim 7, characterized in that The content of the biomacromolecule protein CXCL4 or the secretion-deficient biomacromolecule protein δsp-CXCL4 in the product is not less than 0.5 nM.

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