CN118252937A - Use of CXCR4 receptor antagonists for combating ebola virus infection - Google Patents

Abstract

The invention discloses application of CXCR4 receptor antagonists in resisting Ebola virus infection, CXCR4 has specific interaction with envelope glycoprotein GP of Ebola virus, which is a key cell surface receptor for mediating the adsorption and entry of the Ebola virus into host cells, and the CXCR4 receptor antagonists, CXCR4 antibodies and agents for inhibiting CXCR4 gene and/or CXCR4 protein expression can be used for obviously inhibiting the adsorption and entry of the Ebola virus into target cells, so that the effect of resisting the Ebola virus infection is achieved. The invention provides a new defense means for resisting the virulent infectious diseases.

Description

Application of CXCR4 receptor antagonists in the prevention of Ebola virus infection

Technical Field

The present invention relates to the field of virus infection, and in particular, to the application of a CXCR4 receptor antagonist in resisting Ebola virus infection.

Background technique

Ebola virus disease (EVD) is a severe, hemorrhagic infectious disease caused by Ebolavirus (EBOV) of the Filoviridae family infecting humans and non-human primates. There is currently no effective cure. Therefore, it is crucial to develop highly effective anti-EBOV defense measures.

EBOV has a wide range of tissue cell tropism. Finding the key receptors that can mediate EBOV entry into host cells will help develop new antiviral treatments that target virus adsorption and entry. EBOV envelope glycoprotein GP is located on the surface of virus particles and can mediate virus adsorption and entry by binding to receptors on the surface of corresponding target cells. Reports have shown that the Niemann Pick C1 (NPC1) receptor is a key intracellular receptor for EBOV, which can mediate the fusion process of the virus envelope and endosomal membrane by interacting with the EBOV GP protein in the endosome. In addition, other studies have found that C-type lectin receptors can bind to EBOV non-specifically, thereby mediating EBOV adsorption and entry, but they do not mediate viral internalization into endosomes. Although non-specific receptors such as T cell immunoglobulin mucin 1 (Tim1) can mediate EBOV adsorption and entry, they are only expressed on the surface of a few cells and do not directly interact with EBOV GP. It is difficult for them to independently mediate virus adsorption and entry as virus receptors. Therefore, no key cell surface receptor protein has been found so far that can both specifically interact with the GP protein of EBOV and mediate the adsorption and entry of EBOV.

Summary of the invention

In order to solve the problem that there is no key receptor protein in the prior art that can specifically interact with the GP protein of EBOV and mediate the adsorption and entry of EBOV, and to explore new and efficient anti-EBOV treatment methods, the present invention provides the use of CXCR4 receptor antagonists in anti-Ebola virus infection.

The first object of the present invention is to provide a use of an agent that targets and binds to CXCR4 in the preparation of a product for resisting Ebola virus infection.

The second object of the present invention is to provide the use of a CXCR4 receptor antagonist in the preparation of a product for resisting Ebola virus infection.

The third object of the present invention is to provide the use of CXCR4 antibodies in the preparation of products against Ebola virus infection.

The fourth object of the present invention is to provide the use of an agent for inhibiting the expression of CXCR4 gene and/or CXCR4 protein in the preparation of a product for resisting Ebola virus infection.

A fifth object of the present invention is to provide a biomaterial for inhibiting the expression of CXCR4 gene and/or CXCR4 protein.

The sixth object of the present invention is to provide the use of the biological material in preparing products for resisting Ebola virus infection.

In order to achieve the above object, the present invention is implemented by the following scheme:

CXC chemokine receptor type 4 (CXCR4) is a member of the G protein-coupled receptor (GPCR) superfamily. It can activate downstream signaling pathways and promote tumor cell proliferation and migration by binding to its specific ligand CXC chemokine ligand 12 (CXCL12). In addition, CXCR4 can also act as a co-receptor, binding to the V3 loop of the human immunodeficiency virus (HIV) envelope glycoprotein gp120 through its second extracellular loop, thereby mediating HIV entry into CD4 ⁺ T cells. The present invention proposes for the first time that the cell surface receptor CXCR4 can interact with the EBOV GP protein and can mediate EBOV adsorption and entry into target cells, and accordingly establishes a method for anti-EBOV infection using a receptor antagonist targeting the CXCR4 receptor.

The present invention requests protection for the following contents:

Use of agents that target and bind to CXCR4 in the preparation of products for resisting Ebola virus infection.

Preferably, the agent that targets and binds to CXCR4 comprises an antagonist and/or an antibody of the CXCR4 receptor.

More preferably, the antagonist comprises Motixafortide.

More preferably, the clone number of the antibody is 12G5.

More preferably, the antibody is a monoclonal antibody.

Preferably, the anti-Ebola virus infection comprises inhibiting Ebola virus adsorption and/or entry into target cells.

Application of CXCR4 receptor antagonists in the preparation of products for resisting Ebola virus infection.

Preferably, the antagonist comprises Motixafortide.

Preferably, the anti-Ebola virus infection comprises inhibiting Ebola virus adsorption and/or entry into target cells.

Use of CXCR4 antibodies in the preparation of products against Ebola virus infection.

Preferably, the clone number of the antibody is 12G5.

Preferably, the antibody is a monoclonal antibody.

Preferably, the anti-Ebola virus infection comprises inhibiting Ebola virus adsorption and/or entry into target cells.

Use of an agent for inhibiting the expression of CXCR4 gene and/or CXCR4 protein in the preparation of a product for resisting Ebola virus infection.

Preferably, the reagent is one or more of gRNA, gene editing vector and/or lentivirus, and the gRNA, gene editing vector and lentivirus all target the CXCR4 gene and/or CXCR4 protein.

Preferably, the nucleotide sequences of the gRNA, gene editing vector and target sequence of the lentivirus are as shown in SEQ ID NO.1.

More preferably, the gRNA is synthesized from a nucleic acid molecule having a sequence as shown in SEQ ID NOs. 11 to 12 or a completely complementary sequence of the sequence shown in SEQ ID NOs. 11 to 12.

Preferably, the gene editing vector is a CRISPR gene editing vector containing the gRNA.

More preferably, the gene editing vector uses lenti CRISPR-V2 as a backbone vector.

Preferably, the lentivirus contains the gRNA and/or the gene editing vector.

More preferably, the lentivirus is packaged by the gene editing vector and a helper plasmid.

Further preferably, the helper plasmid includes pVSVg plasmid and psPAX2 plasmid.

Preferably, the anti-Ebola virus infection comprises inhibiting Ebola virus adsorption and/or entry into target cells.

A biomaterial for inhibiting the expression of CXCR4 gene and/or CXCR4 protein, which is any one of the following (1) to (4):

(1) A nucleic acid molecule with a sequence as shown in SEQ ID NOs. 11 to 12 or a completely complementary sequence to the sequence shown in SEQ ID NOs. 11 to 12;

(2) gRNA synthesized from the nucleic acid molecule described in (1);

(3) a gene editing vector containing the gRNA described in (2);

(4) A lentivirus containing the gene editing vector described in (3).

Preferably, the gene editing vector described in (3) is a CRISPR gene editing vector containing the gRNA.

More preferably, the gene editing vector described in (3) uses lenti CRISPR-V2 as a backbone vector.

Preferably, the lentivirus in (4) contains the gRNA and/or the gene editing vector.

More preferably, the lentivirus in (4) is packaged by the gene editing vector and the helper plasmid.

Further preferably, the auxiliary plasmid includes pVSVg plasmid and psPAX2 plasmid.

Application of the biological material in preparing products for resisting Ebola virus infection.

Preferably, the anti-Ebola virus infection comprises inhibiting Ebola virus adsorption and/or entry into target cells.

Use of a reagent that targets and binds to CXCR4 in the preparation of a product that inhibits Ebola virus adsorption and/or entry into target cells.

Preferably, the agent that targets and binds to CXCR4 comprises an antagonist and/or an antibody of the CXCR4 receptor.

More preferably, the antagonist comprises Motixafortide.

More preferably, the clone number of the antibody is 12G5.

More preferably, the antibody is a monoclonal antibody.

Use of a CXCR4 receptor antagonist in the preparation of a product for inhibiting Ebola virus adsorption and/or entry into target cells.

Preferably, the antagonist comprises Motixafortide.

Use of CXCR4 antibodies in the preparation of products for inhibiting Ebola virus adsorption and/or entry into target cells.

Preferably, the clone number of the antibody is 12G5.

Preferably, the antibody is a monoclonal antibody.

Use of an agent for inhibiting the expression of CXCR4 gene and/or CXCR4 protein in the preparation of a product for inhibiting the adsorption and/or entry of Ebola virus into target cells.

Preferably, the reagent is one or more of gRNA, gene editing vector and/or lentivirus, and the gRNA, gene editing vector and lentivirus all target the CXCR4 gene and/or CXCR4 protein.

Preferably, the nucleotide sequences of the gRNA, gene editing vector and target sequence of the lentivirus are as shown in SEQ ID NO.1.

More preferably, the gRNA is synthesized from a nucleic acid molecule having a sequence as shown in SEQ ID NOs. 11 to 12 or a completely complementary sequence of the sequence shown in SEQ ID NOs. 11 to 12.

Preferably, the gene editing vector is a CRISPR gene editing vector containing the gRNA.

More preferably, the gene editing vector uses lenti CRISPR-V2 as a backbone vector.

Preferably, the lentivirus contains the gRNA and/or the gene editing vector.

More preferably, the lentivirus is packaged by the gene editing vector and a helper plasmid.

Further preferably, the helper plasmid includes pVSVg plasmid and psPAX2 plasmid.

The biomaterial is used in preparing a product for inhibiting Ebola virus adsorption and/or entry into target cells.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention discloses for the first time that CXCR4 has a specific interaction with the envelope glycoprotein GP of the Ebola virus, and is a key cell surface receptor that mediates the adsorption and entry of the Ebola virus into host cells. The use of CXCR4 receptor antagonists, CXCR4 antibodies, and reagents that inhibit the expression of the CXCR4 gene and/or CXCR4 protein can significantly inhibit the adsorption and entry of the Ebola virus into target cells, thereby achieving the effect of resisting Ebola virus infection. The present invention provides a new defense means for resisting severe infectious diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the interaction between EBOV GP protein and CXCR4 protein; A is a schematic diagram of the construction of pCAGGS-CXCR4-HA plasmid and pCAGGS-EBOV GP-3×flag plasmid; B is the result of the immunoprecipitation experiment when the detection antibody is an anti-Flag antibody; C is the result of the immunoprecipitation experiment when the detection antibody is an anti-HA antibody.

Figure 2 shows that EBOV GP protein and CXCR4 protein co-localize in cells and promote the internalization of CXCR4 protein; A is a schematic diagram of the construction of pCAGGS-EBOV GP-3×flag-eGFP plasmid; B is the immunofluorescence detection results of EBOV GP-3×flag-eGFP+CXCR4-HA group and 3×flag-eGFP+CXCR4-HA group under laser confocal microscopy; C is the immunofluorescence detection results of EBOV GP-3×flag-eGFP group and 3×flag-eGFP group under laser confocal microscopy; D is the immunofluorescence detection results of 3×flag-eGFP (1.6μg) + CXCR4-HA group, EBOV GP-3×flag-eGFP (100ng) + CXCR4-HA group, EBOV GP-3×flag-eGFP (400ng) + CXCR4-HA group and EBOV under laser confocal microscopy. Immunofluorescence detection results of GP-3×flag-eGFP (1.6μg) + CXCR4-HA group; the scale bar is 5μm.

Figure 3 shows that overexpression of CXCR4 promotes EBOV VLPs adsorption and entry into target cells; A and B are the expression levels of intracellular GP mRNA and VP40 mRNA in the pCAGGS-vector group and pCAGGS-CXCR4-HA group after adsorption of EBOV VLPs for 2 hours at 4°C; C and D are the expression levels of intracellular GP mRNA and VP40 mRNA in the pCAGGS-vector group and pCAGGS-CXCR4-HA group after infection with EBOV VLPs for 3 hours at 37°C; ACTB gene is the internal reference gene; *** indicates P < 0.001, two-sided t test.

Figure 4 shows the knockout effect identification results of the endogenous CXCR4 gene; A is the sequencing results of single cell clones of HEK 293T lenti-V2 and HEK 293TCXCR4-KO; B is the sequencing results of single cell clones of HeLa lenti-V2 and HeLa CXCR4-KO; C is the western blot identification results of single cell clones of HEK 293T lenti-V2 and HEK 293TCXCR4-KO; D is the western blot identification results of single cell clones of HeLa lenti-V2 and HeLa CXCR4-KO.

Figure 5 shows that knocking out endogenous CXCR4 in cells inhibits EBOV VLPs adsorption and entry into target cells; A is the expression level of GP mRNA in HEK 293T lenti-V2 cell line and HEK 293TCXCR4-KO cell line after adsorption or not of EBOV VLPs at 4°C for 2 hours; B is the expression level of GP mRNA in HEK 293T lenti-V2 cell line and HEK 293TCXCR4-KO cell line after infection or not of EBOV VLPs at 37°C for 3 hours; C is the expression level of GP mRNA in HeLalenti-V2 cell line and HeLa CXCR4-KO cell line after adsorption or not of EBOV VLPs at 4°C for 2 hours; D is the expression level of GP mRNA in HeLa lenti-V2 cell line and HeLa CXCR4-KO cell line after infection or not of EBOV VLPs at 37°C for 3 hours. mRNA expression levels; ACTB gene was the internal reference gene; *** indicates P < 0.001, two-sided t test.

Figure 6 Targeted blockade of CXCR4 can inhibit the adsorption and entry of EBOV VLPs into target cells; A and B are the expression levels of GP mRNA in HEK 293T cells and HeLa cells after pretreatment with CXCR4 receptor antagonist for 1 hour, followed by adsorption or non-adsorption of EBOV VLPs at 4°C for 2 hours; C and D are the expression levels of GP mRNA in HEK 293T cells and HeLa cells after pretreatment with CXCR4 receptor antagonist for 1 hour, followed by infection or non-infection with EBOV VLPs at 37°C for 2 hours; E and F are the expression levels of GP mRNA in HEK 293T cells and HeLa cells after pretreatment with CXCR4 monoclonal antibody at a final concentration of 15 μg/mL for 1 hour, followed by adsorption or non-adsorption of EBOV VLPs at 4°C for 2 hours; ACTB gene is the internal reference gene; *** indicates P < 0.001, two-sided t-test.

Detailed ways

The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

Example 1 Interaction between EBOV GP protein and CXCR4 protein

1. Experimental Methods

1. Construction of fusion protein expression plasmid

An HA tag was fused to the carboxyl end of the CXCR4 CDS sequence (SEQ ID NO.1), and a 3×flag tag was fused to the carboxyl end of the EBOV GP sequence (SEQID NO.2). The two fragments were synthesized by gene synthesis, and were respectively denoted as fragment CXCR4-HA and fragment EBOV GP-3×flag. Then, as shown in A in FIG1 , based on the pCAGGS backbone vector, expression plasmids for CXCR4-HA fusion protein (denoted as: pCAGGS-CXCR4-HA) and expression plasmids for EBOV GP-3×flag fusion protein (denoted as: pCAGGS-EBOV GP-3×flag) were constructed.

2. Cell transfection and co-immunoprecipitation

5 μg of pCAGGS-CXCR4-HA plasmid and pCAGGS-EBOV GP-3×flag plasmid were co-transfected into HEK293T cells. After 48 h, the cell pellets were collected and lysed with lysis buffer. A portion of the protein supernatant was used as input, and anti-Flag antibody-coated agarose beads or anti-HA antibody-coated agarose beads were added to the remaining protein supernatant. The homologous IgG antibody was used as a control. Then, the levels of CXCR4-HA and EBOV GP-3×flag fusion proteins in the immune complex were detected by immunoprecipitation. GAPDH protein was used as an internal reference protein. The antibodies used were as follows: anti-Flag antibody (manufacturer: Sigma-Aldrich; catalog number: B3111), anti-HA antibody (manufacturer: MBL; catalog number: M180-3), IgG antibody (manufacturer: Abclonal; catalog number: AC005), GP antibody (manufacturer: Sino Biological; Catalog No.: 40442-T62), CXCR4 (Manufacturer: proteintech; Catalog No.: 60042-1-Ig), GAPDH (Manufacturer: abcam; Catalog No.: ab8245).

2. Experimental Results

As shown in Figure 1 B and C, CXCR4-HA and EBOV GP-3×flag fusion proteins were successfully expressed in HEK 293T cells, and in the immunoprecipitated complex, anti-Flag antibody detected not only EBOV GP-3×flag fusion protein, but also CXCR4-HA fusion protein; similarly, anti-HA antibody also detected CXCR4-HA fusion protein and EBOV GP-3×flag fusion protein, indicating that EBOV GP protein interacts with CXCR4 protein.

Example 2 EBOV GP protein co-localizes with CXCR4 protein in cells and promotes internalization of CXCR4 protein

1. Experimental Methods

1. Construction of fusion protein expression plasmid

As shown in A of Figure 2, based on the pCAGGS-EBOV GP-3×flag plasmid of Example 1, green fluorescent protein (eGFP) was added to the carboxyl terminus of the fragment EBOVGP-3×flag to obtain the fragment EBOV GP-3×flag-eGFP (SEQ ID NO.3), and the expression plasmid of GP-3×flag-eGFP fusion protein was constructed (denoted as: pCAGGS-EBOV GP-3×flag-eGFP plasmid); EBOV GP in pCAGGS-EBOV GP-3×flag-eGFP was removed, and eGFP was added to the carboxyl terminus of 3×flag to obtain the fragment 3×flag-eGFP (SEQ ID NO.4) to construct a control plasmid (denoted as: pCAGGS-3×flag-eGFP plasmid).

2. Cell transfection and cell immunofluorescence

To investigate the subcellular localization of EBOV GP-3×flag-eGFP fusion protein and exogenously expressed CXCR4-HA fusion protein, 2 μg of pCAGGS-CXCR4-HA plasmid was co-transfected with 2 μg of pCAGGS-EBOV GP-3×flag-eGFP plasmid or 2 μg of pCAGGS-3×flag-eGFP plasmid into HEK 293T cells, and the resulting cells were respectively designated as EBOV GP-3×flag-eGFP+CXCR4-HA group and 3×flag-eGFP+CXCR4-HA group.

In order to reflect the actual situation when EBOV infects target cells, the subcellular localization of EBOV GP-3×flag-eGFP and endogenously expressed CXCR4 protein was also investigated. 2 μg pCAGGS-EBOV GP-3×flag-eGFP plasmid and 2 μg pCAGGS-3×flag-eGFP plasmid were separately transfected into HEK 293T cells, and the obtained cells were respectively recorded as: EBOV GP-3×flag-eGFP group and 3×flag-eGFP group.

To further explore whether EBOV GP can promote the internalization of CXCR4 protein, 100 ng, 400 ng and 1.6 μg of pCAGGS-EBOV GP-3×flag-eGFP plasmid were co-transfected with 2 μg of pCAGGS-CXCR4-HA plasmid into HEK 293T cells, respectively. The resulting cells were respectively designated as: EBOV GP-3×flag-eGFP (100 ng) + CXCR4-HA group, EBOV GP-3×flag-eGFP (400 ng) + CXCR4-HA group and EBOV GP-3×flag-eGFP (1.6 μg) + CXCR4-HA group. HEK 293T cells co-transfected with 1.6 μg pCAGGS-3×flag-eGFP plasmid and 2 μg pCAGGS-CXCR4-HA plasmid were used as a control, denoted as 3×flag-eGFP (1.6 μg) + CXCR4-HA group.

After 48 hours of transfection, the cells were fixed with paraformaldehyde and subjected to cell immunofluorescence experiments. The antibodies used were as follows: CXCR4 antibody (manufacturer: proteintech; catalog number: 60042-1-Ig), and DAPI was used to stain the cell nucleus. The fluorescence color development results were observed under a laser confocal microscope.

2. Experimental Results

As shown in Figure 2B, in the 3×flag-eGFP+CXCR4-HA group, the 3×flag-eGFP fusion protein was diffusely distributed in the cells and did not co-localize with the CXCR4-HA protein; in contrast, in the EBOV GP-3×flag-eGFP+CXCR4-HA group, the EBOV GP-3×flag-eGFP fusion protein was mainly distributed in the envelope and in the cytoplasm in a dot-like manner, and co-localized with the CXCR4-HA fusion protein was observed, indicating that the EBOV GP protein co-localized with the exogenously expressed CXCR4-HA fusion protein.

As shown in Figure 2C, in the 3×flag-eGFP group, the 3×flag-eGFP fusion protein was diffusely distributed in the cells and did not co-localize with the CXCR4 protein; in contrast, in the EBOV GP-3×flag-eGFP group, the EBOV GP-3×flag-eGFP fusion protein was mainly distributed in the envelope and in the cytoplasm in a dot-like manner, and co-localized with the CXCR4 protein was observed, indicating that the EBOV GP protein co-localized with the endogenously expressed CXCR4 protein in the cells.

As shown in Figure 2D, in the 3×flag-eGFP (1.6 μg) + CXCR4-HA group, the 3×flag-eGFP fusion protein was diffusely distributed in the cells, while the CXCR4-HA fusion protein was only distributed on the cell membrane surface, and there was no co-localization between the two. In the EBOV GP-3×flag-eGFP (100 ng) + CXCR4-HA group, the EBOV GP-3×flag-eGFP (400 ng) + CXCR4-HA group, and the EBOV GP-3×flag-eGFP (1.6 μg) + CXCR4-HA group, as the transfection dose of EBOV GP-3×flag-eGFP increased, it was seen that the expression position of the GP-3×flag-eGFP fusion protein gradually changed from being mainly distributed on the cell membrane to being mainly distributed in the cytoplasm in a dot-like manner, and consistent with the distribution change, the subcellular distribution of the CXCR4-HA fusion protein also changed from being mainly distributed on the cell membrane to being mainly distributed in the cytoplasm in a dot-like manner, and in the EBOV When the transfection dose of GP-3×flag-eGFP was 400ng and 1.6μg, there was obvious co-localization of GP-3×flag-eGFP and CXCR4-HA fusion protein in the cytoplasm, and the effect was dose-dependent, indicating that EBOV GP can promote the internalization of CXCR4 protein, which is the key factor for CXCR4 to act as a receptor for EBOV-infected target cells.

Example 3 Overexpression of CXCR4 promotes EBOV VLPs adsorption and entry into target cells

1. Experimental Methods

1. Replicable pseudovirus system

In order to explore whether EBOV GP protein can mediate its adsorption and entry into target cells by binding to CXCR4 receptor, a replicable pseudovirus system simulating the entire life cycle of EBOV was established using existing technology (DOI: 10.3791/52381) to synthesize EBOV virus-like particles (referred to as EBOV VLPs). Since the target cells infected by it are not pre-transfected with viral proteins such as NP, VP35, VP30 and L, EBOV VLPs can simulate the adsorption, penetration and transcription of mini-genome of EBOV after infecting target cells.

2. Cell transfection and viral infection

To investigate whether CXCR4 can promote the adsorption of EBOV VLPs and entry into target cells, 2 μg of pCAGGS-CXCR4-HA plasmid and control plasmid (pCAGGS plasmid) were transfected into HEK293T cells separately, and the resulting cells were respectively designated as: pCAGGS-CXCR4-HA group and pCAGGS-vector group.

48 hours after transfection, 600 μL of EBOV VLPs (10 ⁵ to 10 ⁶ copies/mL) were infected and placed at 4°C for 2 hours of adsorption or at 37°C for 3 hours of infection. Total RNA of the two groups of cells was then extracted by TRIzol method and reverse transcribed to obtain cDNA.

3. Real-time fluorescence quantitative PCR

The cDNA of the pCAGGS-CXCR4-HA group and the pCAGGS-vector group obtained in the previous step was used as a template and the primers shown in Table 1 were used to detect the levels of GP mRNA and VP40 mRNA adsorbed on the target cells by real-time fluorescence quantitative PCR.

Table 1 Primers used for real-time fluorescence quantitative PCR

The real-time fluorescence quantitative PCR reaction system was: 2×SYBR Green Mix, 10 μL; Primer F (final concentration 10 umol/L), 1 μL; Primer R (final concentration 10 umol/L), 1 μL; cDNA, 1 μL; H ₂ O, 7 μL.

The reaction conditions of real-time fluorescence quantitative PCR were as follows: (1) 95°C, 5 min, 1 cycle; (2) 95°C, 10 s, 60°C, 30 s, 40 cycles; (3) 95°C, 15 s, 60°C, 30 s, 95°C 15 s, 1 cycle, and the melting curve was drawn.

2. Experimental Results

As shown in A and B in Figure 3, after adsorption of EBOV VLPs at 4°C for 2 h, the intracellular GP mRNA and VP40 mRNA levels in the pCAGGS-CXCR4-HA group were significantly increased compared with the pCAGGS-vector group, indicating that overexpression of CXCR4 can promote the adsorption of EBOV VLPs on the surface of target cells.

As shown in Figure 3C and D, after infection with EBOV VLPs at 37°C for 3 h, the levels of GP mRNA and VP40 mRNA in the pCAGGS-CXCR4-HA group were significantly increased compared with the pCAGGS-vector group, indicating that overexpression of CXCR4 promotes the entry of EBOV VLPs into target cells.

Example 4 Knockout of endogenous CXCR4 inhibits EBOV VLPs adsorption and entry into target cells

1. Experimental Methods

1. Construction of cell lines with endogenous CXCR4 gene knockout

Endogenous CXCR4 gene knockout cell lines (HEK293T CXCR4-KO and HeLa CXCR4-KO) were constructed in HEK 293T and HeLa cell lines, respectively. The specific methods are as follows:

(1) Construction of gene editing plasmid

Synthesize gRNA targeting the CXCR4 CDS region, the top primer sequence of the gRNA is 5'-CACCGTCTTCTGGTAACCCATGACC-3' (SEQ ID NO.11); the bottom primer sequence is 5'-AAACGGTCATGGGTTACCAGAAGAC-3' (SEQ ID NO.12). 5μL top primer and 5μL bottom primer were boiled at 100℃ for 10min, and then naturally cooled to room temperature to obtain annealing products. The lenti CRISPR-V2 backbone plasmid was digested with BsmBI, placed at 37℃ for 2h, and then agarose gel electrophoresis and gel recovery were performed to obtain the digested lenti CRISPR-V2 linearized DNA fragment. The annealing product was ligated with the linearized lenti CRISPR-V2 fragment overnight by T4 ligase to obtain a gene editing plasmid for knocking out the CXCR4 gene, which was recorded as lenti-CRISPR-CXCR4 plasmid.

(2) Lentiviral packaging and infection

The lenti-CRISPR-CXCR4 plasmid, auxiliary plasmid pVSVg plasmid and psPAX2 plasmid were transfected into HEK 293T cells and HeLa cells at a mass ratio of 6:3:4.5, and polybrene with a final concentration of 10μg/mL was added to promote infection. The medium was changed 24 hours after transfection, and after another 24 hours, puromycin with a final concentration of 2μg/mL was added for selection until all cells in the uninfected group died, and then the puromycin concentration was reduced to 1μg/mL. The cells were then plated into 96-well plates to screen single-cell clones, and the single-cell clones were sequenced and identified by western blot (the antibodies used were as follows: CXCR4 antibody (manufacturer: proteintech; catalog number: 60042-1-Ig), α-tubulin protein was the internal reference protein (manufacturer: MBL, catalog number: PM054)). Cells with expected identification results were the knockout cell lines of the endogenous CXCR4 gene, which were recorded as HEK 293TCXCR4-KO and HeLaCXCR4-KO, respectively.

Following the same method, the lenti CRISPR-V2 backbone plasmid, auxiliary plasmids pVSVg plasmid and psPAX2 plasmid were transfected into HEK 293T cells and HeLa cells to construct control cell lines, which were named HEK 293T lenti-V2 and HeLa lenti-V2, respectively.

As shown in A and B in Figure 4, the sequencing results showed that compared with their respective control cell lines, the CDS region fragment of the CXCR4 gene was missing in HEK 293TC CXCR4-KO and HeLa CXCR4-KO. As shown in C and D in Figure 4, the western blot results showed that compared with their respective control cell lines, CXCR4 protein was basically not expressed in HEK 293T CXCR4-KO and HeLa CXCR4-KO. The above results indicate that the knockout cell line of the endogenous CXCR4 gene was successfully constructed.

2. Viral infection

To further confirm that CXCR4 mediates EBOV VLPs adsorption and entry into target cells, knockout cell lines (HEK 293TCXCR4-KO and HeLa CXCR4-KO) and control cell lines (HEK 293T lenti-V2 and HeLa lenti-V2) were infected with 600 μL EBOV VLPs (10 ⁵ ～10 ⁶ copies/mL), and each cell line (CTRL) not infected with EBOV VLPs was used as a control, and placed at 4°C for adsorption for 2 hours or at 37°C for infection for 3 hours. Total RNA of each cell was then extracted by TRIzol method and reverse transcribed to obtain cDNA.

3. Real-time fluorescence quantitative PCR

The cDNA obtained in the previous step was used as a template and the primers shown in Table 1 were used to detect the level of GP mRNA adsorbed on the target cells by real-time fluorescence quantitative PCR. The PCR reaction system and PCR reaction conditions were the same as those in Example 3.

2. Experimental Results

As shown in A and C of Figure 5 , after adsorption of EBOV VLPs at 4°C for 2 h, the GP mRNA levels in HEK 293T cells and HeLa cells after CXCR4 gene knockout were significantly reduced compared with the control cell lines.

As shown in Figure 5, B and D, after infection with EBOV VLPs at 37°C for 3 h, the GP mRNA levels in HEK 293T and HeLa cells after CXCR4 gene knockout were significantly reduced compared with the control cell lines.

The above results indicate that knocking out the intracellular CXCR4 gene inhibits the adsorption and entry of EBOV VLPs into target cells.

Example 5 CXCR4 receptor antagonists inhibit EBOV VLPs adsorption and entry into target cells

1. Experimental Methods

1. Receptor antagonist treatment and cell infection

HEK 293T cells and HeLa cells were pretreated with CXCR4 receptor antagonist Motixafortide (HY-P0171, MedChemExpress) at a final concentration of 20 μM for 1 h (DMSO was used as solvent control), and then infected with 600 μL EBOV VLPs (10 ⁵ ～10 ⁶ copies/mL). Each cell line (CTRL) not infected with EBOV VLPs was used as a control and placed at 4°C for adsorption for 2 h or at 37°C for infection for 3 h. Total RNA of each cell was then extracted by TRIzol method and reverse transcribed to obtain cDNA.

2. Monoclonal Antibody Treatment and Cell Infection

HEK 293T cells were pretreated with CXCR4-specific antibody 12G5 (16-9999-81, Invitrogen) at a final concentration of 15 μg/mL for 1 h, and HeLa cells were pretreated with CXCR4-specific antibody 12G5 at a final concentration of 20 μg/mL for 1 h, and then infected with 600 μL EBOV VLPs (10 ⁵ ～10 ⁶ copies/mL). Each cell line (CTRL) not infected with EBOV VLPs was used as a control and placed at 4°C for adsorption for 2 h. Total RNA of each cell was then extracted by TRIzol method and reverse transcribed to obtain cDNA.

3. Real-time fluorescence quantitative PCR

The cDNA obtained in the previous step was used as a template and the primers shown in Table 1 were used to detect the level of GP mRNA adsorbed on the target cells by real-time fluorescence quantitative PCR. The PCR reaction system and PCR reaction conditions were the same as those in Example 3.

2. Experimental Results

As shown in A and B in Figure 6, after adsorption of EBOV VLPs at 4°C for 2 h, the GP mRNA levels in HEK 293T cells and HeLa cells treated with the CXCR4 receptor antagonist Motixafortide were significantly reduced, indicating that the CXCR4 receptor antagonist inhibited the adsorption of EBOV VLPs on the target cell surface.

As shown in Figure 6C and D, after infection with EBOV VLPs at 37°C for 3 h, the GP mRNA levels in HEK 293T cells and HeLa cells treated with the CXCR4 receptor antagonist Motixafortide were significantly reduced, indicating that the CXCR4 receptor antagonist inhibited the entry of EBOV VLPs into target cells.

As shown in Figure 6E and F, after 2h of adsorption of EBOV VLPs at 4°C, the GP mRNA levels in HEK 293T cells and HeLa cells treated with CXCR4 monoclonal antibody 12G5 were significantly reduced, indicating that antibodies targeting CXCR4 receptors can block CXCR4-mediated EBOV VLPs adsorption and entry into target cells.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims (10)

1. Use of an agent that targets binding to CXCR4 in the preparation of a product against ebola virus infection.

2. The use of claim 1, wherein the agent that targets binding to CXCR4 comprises an antagonist of CXCR4 receptor and/or an antibody.

Use of an antagonist of cxcr4 receptor for the preparation of a product against ebola virus infection.

Use of an antibody to cxcr4 for the preparation of a product against ebola virus infection.

5. Use of an agent that inhibits CXCR4 gene and/or CXCR4 protein expression in the preparation of a product against ebola virus infection.

6. The use according to claim 5, wherein the agent is one or more of a gRNA, a gene editing vector and/or a lentivirus, each targeting a CXCR4 gene and/or a CXCR4 protein.

7. The use according to claim 6, wherein the nucleotide sequences of the target sequences of the gRNA, gene editing vector and lentivirus are shown in SEQ ID NO. 1.

8. A biological material for inhibiting expression of CXCR4 gene and/or CXCR4 protein, which is any one of the following (1) to (4):

(1) Nucleic acid molecules with sequences shown as SEQ ID NO. 11-12 or the complete complementary sequences of the sequences shown as SEQ ID NO. 11-12;

(2) A gRNA synthesized from the nucleic acid molecule of (1);

(3) A gene editing vector comprising the gRNA of (2);

(4) A lentivirus comprising the gene editing vector as described in (3).

9. Use of the biomaterial of claim 8 for the preparation of an ebola virus infection resistant product.

10. The use according to any one of claims 1 to 7 or claim 9, wherein said anti-ebola virus infection comprises inhibiting ebola virus adsorption and/or entry into a target cell.