CN118252937B - Use of CXCR4 receptor antagonists for combating ebola virus infection - Google Patents
Use of CXCR4 receptor antagonists for combating ebola virus infection Download PDFInfo
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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
Technical Field
The present invention relates to the field of viral infections, in particular to the use of antagonists of the CXCR4 receptor for combating Ebola virus infections.
Background
Ebola Virus (EVD) is a virulent, hemorrhagic infectious disease caused by infection of humans and non-human primates with Ebola virus (EBOV) of the family filoviridae, and no effective cure is currently available. Therefore, it is important to develop a highly effective defense against EBOV.
The EBOV has wide tissue cell tropism, and searching for a key receptor which can mediate the EBOV to enter host cells can help to develop a novel antiviral treatment means targeting the viral adsorption and entry links. The EBOV envelope glycoprotein GP is located on the surface of viral particles and mediates viral adsorption and entry events by binding to the receptor on the surface of the corresponding target cell. It has been reported that NIEMANN PICK C (NPC 1) receptor is a key intracellular receptor for EBOV and can mediate the viral envelope and endosomal membrane fusion process in endosomes by interacting with the EBOV GP protein. In addition, it has been found that the C-type lectin receptor can bind non-specifically to EBOV, thereby mediating EBOV adsorption and entry, but it does not mediate viral internalization into endosomes. While non-specific receptors such as T cell immunoglobulin mucin 1 (Tim 1) can mediate adsorption and entry of EBOV, but are only expressed on a few cell surfaces and do not directly interact with EBOV GP, and are difficult to be used as viral receptors to independently mediate adsorption and entry links of viruses. Therefore, it has not been found at present that it is capable of specifically interacting with the GP protein of EBOV and mediating the adsorption and entry of EBOV to the key cell surface receptor protein.
Disclosure of Invention
In order to solve the problem that the prior art has no interaction between specificity and GP protein of the EBOV and can mediate the adsorption and the entering of the EBOV, the invention provides the application of CXCR4 receptor antagonist in resisting Ebola virus infection for exploring a novel and efficient anti-EBOV treatment means.
It is a first object of the present invention to provide the use of an agent that targets binding to CXCR4 in the preparation of a product resistant to ebola virus infection.
A second object of the present invention is to provide the use of an antagonist of CXCR4 receptor for the preparation of a product against ebola virus infection.
A third object of the present invention is to provide the use of an antibody to CXCR4 in the preparation of a product against ebola virus infection.
A fourth object of the present invention is to provide the use of an agent that inhibits CXCR4 gene and/or CXCR4 protein expression in the preparation of a product against ebola virus infection.
A fifth object of the present invention is to provide a biological material that inhibits CXCR4 gene and/or CXCR4 protein expression.
A sixth object of the present invention is to provide the use of said biological material for the preparation of a product resistant to ebola virus infection.
In order to achieve the above object, the present invention is realized by the following means:
CXC chemokine receptor 4 (CXC chemokine receptor type, cxcr 4) is one of the G protein-coupled receptor (GPCR) superfamily members that activates downstream signaling pathways by binding to its specific ligand CXC chemokine ligand 12 (CXC chemokine ligand, cxcr 12), promoting tumor cell proliferation and migration. Furthermore, CXCR4 can also act as a co-receptor, via which the second extracellular loop binds to the V3 loop of the human immunodeficiency virus (Human immunodeficiency virus, HIV) envelope glycoprotein gp120, thereby mediating HIV entry into CD4 + T cells. The invention provides for the first time that the cell surface receptor CXCR4 can interact with the EBOV GP protein and can mediate the absorption and entry of the EBOV into target cells, and accordingly establishes a method for resisting EBOV infection by using a receptor antagonist to target the CXCR4 receptor.
The invention claims the following:
use of an agent that targets binding to CXCR4 in the preparation of a product against ebola virus infection.
Preferably, the agent that targets binding to CXCR4 comprises an antagonist and/or an antibody to the CXCR4 receptor.
More preferably, the antagonist comprises Motixafortide.
More preferably, the antibody has clone number 12G5.
More preferably, the antibody is a monoclonal antibody.
Preferably, the anti-ebola virus infection comprises inhibiting ebola virus adsorption and/or entry into a target cell.
Use of an antagonist of the CXCR4 receptor for the preparation of a product against ebola virus infection.
Preferably, the antagonist comprises Motixafortide.
Preferably, the anti-ebola virus infection comprises inhibiting ebola virus adsorption and/or entry into a target cell.
Use of an antibody to CXCR4 in the preparation of a product against ebola virus infection.
Preferably, the antibody has a clone number of 12G5.
Preferably, the antibody is a monoclonal antibody.
Preferably, the anti-ebola virus infection comprises inhibiting ebola virus adsorption and/or entry into a target cell.
Use of an agent that inhibits CXCR4 gene and/or CXCR4 protein expression in the preparation of a product against ebola virus infection.
Preferably, 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.
Preferably, the nucleotide sequences of the target sequences of the gRNA, the gene editing vector and the lentivirus are 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 NO. 11-12 or a completely complementary sequence of the sequences shown in SEQ ID NO. 11-12.
Preferably, the gene editing vector is a CRISPR gene editing vector comprising 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 from the gene editing vector and helper plasmid.
Further preferably, the helper plasmids include pVSVg plasmid and psPAX plasmid.
Preferably, the anti-ebola virus infection comprises inhibiting ebola virus adsorption and/or entry into a target cell.
A biological material that inhibits expression of a CXCR4 gene and/or a 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).
Preferably, the gene editing vector in (3) is a CRISPR gene editing vector comprising the gRNA.
More preferably, the gene editing vector of (3) uses LENTI CRISPR-V2 as a backbone vector.
Preferably, the lentivirus of (4) contains the gRNA and/or the gene editing vector.
More preferably, the lentivirus of (4) is packaged from the gene editing vector and helper plasmid.
Further preferably, the helper plasmids include pVSVg plasmid and psPAX plasmid.
The 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 a target cell.
Use of an agent that targets binding CXCR4 in the preparation of a product that inhibits ebola virus adsorption and/or entry into a target cell.
Preferably, the agent that targets binding to CXCR4 comprises an antagonist and/or an antibody to the CXCR4 receptor.
More preferably, the antagonist comprises Motixafortide.
More preferably, the antibody has clone number 12G5.
More preferably, the antibody is a monoclonal antibody.
Use of an antagonist of the CXCR4 receptor for the preparation of a product for inhibiting the adsorption and/or entry of ebola virus into a target cell.
Preferably, the antagonist comprises Motixafortide.
Use of an antibody to CXCR4 in the preparation of a product which inhibits ebola virus adsorption and/or entry into a target cell.
Preferably, the antibody has a clone number of 12G5.
Preferably, the antibody is a monoclonal antibody.
Use of an agent that inhibits CXCR4 gene and/or CXCR4 protein expression in the preparation of a product that inhibits ebola virus adsorption and/or entry into a target cell.
Preferably, 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.
Preferably, the nucleotide sequences of the target sequences of the gRNA, the gene editing vector and the lentivirus are 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 NO. 11-12 or a completely complementary sequence of the sequences shown in SEQ ID NO. 11-12.
Preferably, the gene editing vector is a CRISPR gene editing vector comprising 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 from the gene editing vector and helper plasmid.
Further preferably, the helper plasmids include pVSVg plasmid and psPAX plasmid.
The use of said biological material for the preparation of a product for inhibiting the adsorption and/or entry of ebola virus into a target cell.
Compared with the prior art, the invention has the following beneficial effects:
The invention discloses that CXCR4 and GP have specific interaction with each other, which is a key cell surface receptor for mediating the adsorption and entry of the Ebola virus into host cells, and the CXCR4 receptor antagonist, the CXCR4 antibody and the reagent for inhibiting the expression of CXCR4 gene and/or CXCR4 protein can obviously inhibit the adsorption and entry of the Ebola virus into target cells, thereby achieving the effect of resisting the infection of the Ebola virus. The invention provides a new defense means for resisting the virulent infectious diseases.
Drawings
FIG. 1 shows interaction of EBOV GP protein with CXCR4 protein; a is a schematic diagram for constructing pCAGGS-CXCR4-HA plasmid and pCAGGS-EBOV GP-3 xflag plasmid; b is the result of an immune coprecipitation experiment in which the detection antibody is an anti-Flag antibody; c is the result of co-immunoprecipitation experiment in which the detection antibody is an anti-HA antibody.
FIG. 2 is a schematic illustration of the intracellular co-localization of the EBOV GP protein and the CXCR4 protein and promotion of the internalization of the CXCR4 protein; a is a schematic diagram for constructing pCAGGS-EBOV GP-3 xflag-eGFP plasmid; b is an immunofluorescence detection result of the EBOV GP-3 xflag-eGFP+CXCR4-HA group and the 3 xflag-eGFP+CXCR4-HA group under a laser confocal microscope; c is the immunofluorescence detection result of the EBOV GP-3 xflag-eGFP group and the 3 xflag-eGFP group under the laser confocal microscope; d is the immunofluorescence detection result of 3 Xflag-eGFP (1.6 mug) +CXCR4-HA group, EBOV GP-3 Xflag-eGFP (100 ng) +CXCR4-HA group, EBOV GP-3 Xflag-eGFP (400 ng) +CXCR4-HA group and EBOV GP-3 Xflag-eGFP (1.6 mug) +CXCR4-HA group under a laser confocal microscope; the scale is 5. Mu.m.
FIG. 3 is a graph showing that overexpression of CXCR4 promotes adsorption of EBOV VLPs into target cells; the expression levels of intracellular GP mRNA and VP40mRNA of the pCAGGS-vector group and the pCAGGS-CXCR4-HA group after the adsorption of EBOV VLPs at 4 ℃ for 2h are respectively shown in A and B; c and D are the expression levels of intracellular GP mRNA and VP40mRNA of the post-pCAGGS-vector group and the pCAGGS-CXCR4-HA group after infection of EBOV VLPs at 37℃for 3h, respectively; the ACTB gene is a reference gene; * P <0.001, double sided t-test.
FIG. 4 is a graph depicting the results of the identification of the knockout effect of endogenous CXCR4 gene; a is the single cell clone sequencing result of HEK 293T lenti-V2 and HEK 293T CXCR4-KO; b is HELA LENTI-V2 and HeLa CXCR 4-KO; c is the identification result of single-cell clone western blot of HEK 293T lenti-V2 and HEK 293 TCXCR-KO; DHELA LENTI-V2 and HeLa CXCR 4-KO.
FIG. 5 shows that knockout of endogenous CXCR4 of a cell inhibits adsorption of EBOV VLPs into a target cell; the expression level of GP mRNA in HEK 293T lenti-V2 cell line and HEK 293 TCXCR-KO cell line after 2h of EBOV VLPs is absorbed or not absorbed at 4 ℃; b is the expression level of GP mRNA in the EBOV VLPs3h, HEK 293T lenti-V2 cell line and HEK 293T CXCR4-KO cell line infected or not infected at 37 ℃; the expression level of GP mRNA in HELA LENTI-V2 cell line and HeLa CXCR4-KO cell line after 2h of EBOV VLPs is adsorbed or not adsorbed at 4 ℃; d is the expression level of GP mRNA in the EBOV VLPs3h, heLa lenti-V2 cell line and HeLa CXCR4-KO cell line, infected or not infected at 37 ℃; the ACTB gene is a reference gene; * P <0.001, double sided t-test.
FIG. 6 targeted blocking of CXCR4 inhibits adsorption of EBOV VLPs into target cells; a and B are the expression level of GP mRNA in each cell after pretreatment of HEK 293T cells and HeLa cells with CXCR4 receptor antagonists for 1h and then adsorption or non-adsorption of EBOV VLPs 2h at 4 ℃; c and D are expression levels of GP mRNA in HEK 293T cells and HeLa cells after pretreatment with CXCR4 receptor antagonist for 1h, respectively, and then infected or not infected with EBOV VLPs 2h at 37 ℃; e and F are respectively the expression level of GP mRNA in HEK 293T cells pretreated by CXCR4 monoclonal antibody with a final concentration of 15 mug/mL and HELa cells pretreated by CXCR4 monoclonal antibody with a final concentration of 20 mug/mL for 1h, and then EBOV VLPs 2h is adsorbed or not adsorbed at 4 ℃; the ACTB gene is a reference gene; * P <0.001, double sided t-test.
Detailed Description
The invention will be further described in detail with reference to the drawings and specific examples, which are given solely for the purpose of illustration and are not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
Example 1 interaction of EBOV GP protein with CXCR4 protein
1. Experimental method
1. Construction of fusion protein expression plasmid
1 HA tag is fused at the carboxyl end of CXCR4 CDS sequence (SEQ ID NO. 1), 3 xflag tag is fused at the carboxyl end of EBOV GP sequence (SEQ ID NO. 2), the two fragments are synthesized by a gene synthesis mode, and the fragments are sequentially marked as fragments CXCR4-HA and fragments EBOV GP-3 xflag. Then, as shown in A in FIG. 1, on the basis of the pCAGGS skeleton vector, expression plasmids of CXCR4-HA fusion protein (named: pCAGGS-CXCR 4-HA) and expression plasmids of EBOV GP-3 Xflag fusion protein (named: pCAGGS-EBOV GP-3 Xflag) were constructed, respectively.
2. Cell transfection and co-immunoprecipitation
5 Μg of each of the pCAGGS-CXCR4-HA plasmid and the pCAGGS-EBOV GP-3 Xflag plasmid was co-transfected into HEK 293T cells, and after 48 hours, cell pellet was collected, and lysed with lysate, wherein a part of protein supernatant was used as input, agarose beads coated with anti-Flag antibody or agarose beads coated with anti-HA antibody were added to the remaining protein supernatant, and homologous IgG antibody was used as a control, and then the levels of CXCR4-HA and EBOV GP-3 Xflag fusion proteins in the immune complex were detected by the method of immunoprecipitation, and GAPDH protein was used as an internal reference protein as follows: anti-Flag antibody (manufacturer: sigma-Aldrich; cat# B3111), anti-HA antibody (manufacturer: MBL; cat# M180-3), igG antibody (manufacturer: abclonal; cat# AC 005), GP antibody (manufacturer: sino Biological; cat# 40442-T62), CXCR4 (manufacturer: proteintech; cat# 60042-1-Ig), GAPDH (manufacturer: abcam; cat# ab 8245).
2. Experimental results
As shown in FIGS. 1B and C, CXCR4-HA and EBOV GP-3 Xflag fusion proteins were both successfully expressed in HEK 293T cells, respectively, and not only was EBOV GP-3 Xflag fusion protein detected using anti-Flag antibodies, but also CXCR4-HA fusion protein was detected in immunoprecipitated complexes; similarly, CXCR4-HA fusion proteins and EBOV GP-3 Xflag fusion proteins were also detected using anti-HA antibodies. Indicating interaction between EBOV GP protein and CXCR4 protein.
Example 2EBOV GP protein co-localizes with CXCR4 protein in cells and promotes internalization of CXCR4 protein
1. Experimental method
1. Construction of fusion protein expression plasmid
As shown in FIG. 2A, on the basis of the pCAGGS-EBOV GP-3 xflag plasmid of example 1, a green fluorescent protein (eGFP) was added to the carboxyl end of the fragment EBOV GP-3 xflag to obtain a fragment EBOV GP-3 xflag-eGFP (SEQ ID NO. 3), and an expression plasmid (named: pCAGGS-EBOV GP-3 xflag-eGFP plasmid) was constructed to obtain a GP-3 xflag-eGFP fusion protein; the EBOV GP in pCAGGS-EBOV GP-3 Xflag-eGFP was removed, and eGFP was added to the carboxyl end of 3 Xflag to obtain fragment 3 Xflag-eGFP (SEQ ID NO. 4) to construct a control plasmid (designated as pCAGGS-3 Xflag-eGFP plasmid).
2. Cell transfection and cellular immunofluorescence
To investigate the subcellular localization of the EBOV GP-3 Xflag-eGFP fusion protein with exogenously expressed CXCR4-HA fusion protein, 2. Mu.g of the pCAGGS-CXCR4-HA plasmid was co-transfected with 2. Mu.g of the pCAGGS-EBOV GP-3 Xflag-eGFP plasmid or 2. Mu.g of the pCAGGS-3 Xflag-eGFP plasmid into HEK 293T cells, which were in turn scored: EBOV GP-3 Xflag-eGFP+CXCR4-HA group and 3 Xflag-eGFP+CXCR4-HA group.
To reflect the reality of EBOV infection of target cells, subcellular localization of EBOV GP-3 Xflag-eGFP with CXCR4 protein expressed endogenously by the cells was also explored, 2. Mu.g of pCAGGS-EBOV GP-3 Xflag-eGFP plasmid and 2. Mu.g of pCAGGS-3 Xflag-eGFP plasmid were separately transfected into HEK 293T cells, respectively, and the resulting cells were sequentially scored: EBOV GP-3 Xflag-eGFP group and 3 Xflag-eGFP group.
To further investigate whether EBOV GP promoted internalization of CXCR4 protein, 100ng, 400ng and 1.6 μg of pCAGGS-EBOV GP-3 xflag-eGFP plasmid were co-transfected with 2 μ gpCAGGS-CXCR4-HA plasmid into HEK 293T cells, respectively, and the resulting cells were in turn: EBOV GP-3 Xflag-eGFP (100 ng) +CXCR4-HA group, EBOV GP-3 Xflag-eGFP (400 ng) +CXCR4-HA group, and EBOV GP-3 Xflag-eGFP (1.6 μg) +CXCR4-HA group. HEK 293T cells co-transformed with 1.6. Mu.g of pCAGGS-3 Xflag-eGFP plasmid and 2. Mu.g of pCAGGS-CXCR4-HA plasmid were used as controls and were designated as 3 Xflag-eGFP (1.6. Mu.g) +CXCR4-HA group.
After 48h transfection of each of the above cells, cells were fixed using paraformaldehyde, and a cell immunofluorescence experiment was performed using the following antibodies: CXCR4 antibody (manufacturer: proteintech; cat# 60042-1-Ig) was stained with DAPI for nuclei. The fluorescent color development results were observed under a confocal laser microscope.
2. Experimental results
As shown in B in fig. 2, in the 3×flag-egfp+cxcr4-HA group, the 3×flag-eGFP fusion protein is dispersed in the cell and is not co-localized with the CXCR4-HA protein; in contrast, in the EBOV GP-3 xflag-eGFP+CXCR4-HA group, the EBOV GP-3 xflag-eGFP fusion proteins are mainly distributed in the envelope and intracytoplasmic punctiform, and can be co-localized with the CXCR4-HA fusion proteins. Indicating that the EBOV GP protein is co-localized with exogenously expressed CXCR4-HA fusion proteins.
As shown in C in fig. 2, in the 3×flag-eGFP group, 3×flag-eGFP fusion proteins are dispersed in cells and do not co-localize with CXCR4 protein; in contrast, in the EBOV GP-3 xflag-eGFP group, the EBOV GP-3 xflag-eGFP fusion proteins were predominantly envelope and intracytoplasmic punctiform, and co-localization with CXCR4 proteins was seen. Indicating that the EBOV GP protein is co-localized with CXCR4 protein expressed endogenously by the cell.
As shown in D in fig. 2,3×flag-eGFP (1.6 μg) +cxcr4-HA group, 3×flag-eGFP fusion protein is dispersed in cells, whereas CXCR4-HA fusion protein is only distributed on cell membrane surface, and co-localization phenomenon is not seen in both; whereas in the EBOV GP-3 xflag-eGFP (100 ng) +CXCR4-HA group, the EBOV GP-3 xflag-eGFP (400 ng) +CXCR4-HA group and the EBOV GP-3 xflag-eGFP (1.6 μg) +CXCR4-HA group, as the dose of the EBOV GP-3 xflag-eGFP transfection increases, it was seen that the expression position of the GP-3 xflag-eGFP fusion protein gradually changed from a cell membrane distribution to a cytoplasmic punctiform distribution, and consistent with the change in the distribution, the subcellular distribution of the CXCR4-HA fusion protein also changed from a cell membrane distribution to a cytoplasmic punctiform distribution, and that there was a significant co-localization of the GP-3 xflag-eGFP and the 4-HA fusion protein in the cytoplasm with dose dependency in the presence of dose-1.6 μg transfection doses of the EBOV GP-3 xflag-eGFP at 400ng and 1.6 μg. EBOV GP was shown to promote internalization of CXCR4 protein, which is a key factor in the infection of target cell receptors by EBOV for CXCR 4.
Example 3 overexpression of CXCR4 promotes adsorption of EBOV VLPs into target cells
1. Experimental method
1. Replication-competent pseudovirus system
To investigate whether the EBOV GP protein can mediate its adsorption and entry into target cells by binding to the CXCR4 receptor, EBOV virus-like particles (noted EBOV VLPs) were synthesized using a replicable pseudoviral system established by the prior art (DOI: 10.3791/52381) that mimics the complete life cycle of EBOV. Since the target cells infected by the EBOV VLPs are not pre-transfected with the NP, VP35, VP30, L and other proteins of the virus, the adsorption, penetration and minigenome (mini-genome) transcription process of the EBOV can be simulated after the target cells are infected by the EBOV VLPs.
2. Cell transfection and viral infection
To investigate whether CXCR4 promotes the adsorption and entry of EBOV VLPs into target cells, the 2 μ gpCAGGS-CXCR4-HA plasmid and the control plasmid (pCAGGS plasmid) were transfected into HEK293T cells separately, respectively, and the resulting cells were in turn: the pCAGGS-CXCR4-HA group and the pCAGGS-vector group.
After 48h of transfection 600. Mu.L of EBOV VLPs (10 5～106 copies/mL) were infected, either at 4℃for 2h of adsorption or at 37℃for 3h of infection. Then extracting total RNA of the two groups of cells by TRIzol method, and obtaining cDNA by reverse transcription.
3. Real-time fluorescent quantitative PCR
The cDNAs of the pCAGGS-CXCR4-HA group and the pCAGGS-vector group obtained in the above step were used as templates, and the levels of GP mRNA and VP40 mRNA adsorbed on target cells were detected by a real-time fluorescent quantitative PCR method using the primers shown in Table 1.
TABLE 1 primers for real-time fluorescent quantitative PCR
The real-time fluorescent quantitative PCR reaction system is as follows: 2 XSYBR Green Mix, 10. Mu.L; primer F (final concentration 10 umol/L), 1. Mu.L; primer R (final concentration 10 umol/L), 1. Mu.L; cDNA, 1. Mu.L; h 2 O, 7. Mu.L.
The real-time fluorescent quantitative PCR reaction conditions are as follows: (1) 95 ℃,5min,1cycle; (2) 95 ℃,10s,60 ℃,30s,40cycles; (3) Dissolution curves were drawn at 95℃for 15s,60℃for 30s, and 95℃for 15s,1 cycle.
2. Experimental results
As shown in FIGS. 3A and B, after adsorption of EBOV VLPs at 4℃for 2h, the intracellular GP mRNA and VP40 mRNA levels were significantly increased in the pCAGGS-CXCR4-HA group compared to the pCAGGS-vector group. It was shown that overexpression of CXCR4 promotes adsorption of EBOV VLPs on the target cell surface.
As shown in FIGS. 3C and D, the intracellular GP mRNA and VP40 mRNA levels of the pCAGGS-CXCR4-HA group were significantly increased as compared to the pCAGGS-vector group after infection with EBOV VLPs for 3h at 37 ℃. It was shown that overexpression of CXCR4 promotes entry of EBOV VLPs into target cells.
Example 4 knockout of endogenous CXCR4 from cells inhibits adsorption of EBOV VLPs into target cells
1. Experimental method
1. Construction of endogenous CXCR4 Gene knockout cell line
Endogenous CXCR4 knockout cell lines (HEK 293T CXCR4-KO and HeLa CXCR4-KO) were constructed in two cell lines, HEK 293T and HeLa, respectively, by the following methods:
(1) Construction of Gene editing plasmid
Synthesizing gRNA targeting CXCR4 CDS region, wherein 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). 5. Mu.L of top primer and 5. Mu.L of bottom primer were boiled at 100℃for 10min, and then naturally cooled to room temperature to give an annealed product. The LENTI CRISPR-V2 skeleton plasmid is digested with BsmBI, placed at 37 ℃ for 2 hours, and then subjected to agarose gel electrophoresis and gel recovery to obtain the digested LENTI CRISPR-V2 linearized DNA fragment. The annealed product was ligated overnight with the linearized LENTI CRISPR-V2 fragment by T4 ligase to obtain a gene editing plasmid for knocking out CXCR4 gene, designated lenti-CRISPR-CXCR4 plasmid.
(2) Lentivirus packaging and infection
The lenti-CRISPR-CXCR4 plasmid was combined with helper plasmid pVSVg plasmid and psPAX plasmid according to 6:3:4.5 mass ratio into HEK 293T cells and HeLa cells, respectively, and adding polybrene to a final concentration of 10. Mu.g/mL to promote infection. After 24h of transfection, the solution is changed, after 24h of further treatment, puromycin with a final concentration of 2 mug/mL is added for screening until the cells of the uninfected group die and the puromycin concentration is reduced to 1 mug/mL. Then spreading the cells to a 96-well plate to screen single cell clones, sequencing the single cell clones, and performing western blot identification (the antibodies are as follows: CXCR4 antibody (manufacturer: proteintech; cat# 60042-1-Ig), alpha-tubulin protein is an internal reference protein (manufacturer: MBL, cat# PM 054)), and the identification results are the expected knockout cell lines of endogenous CXCR4 genes, which are respectively marked as HEK 293TCXCR4-KO and HeLa CXCR4-KO.
The LENTI CRISPR-V2 backbone plasmid, helper plasmid pVSVg plasmid and psPAX plasmid were transfected into HEK 293T cells and HeLa cells in the same manner to construct control cell lines, designated HEK 293T lenti-V2 and HELA LENTI-V2, respectively.
As shown in a and B in fig. 4, the sequencing results showed that CDS region fragments of CXCR4 gene had been deleted in both HEK 293TCXCR4-KO and HeLa CXCR4-KO compared to the respective control cell lines. As shown by C and D in FIG. 4, the western blot detection results showed that CXCR4-KO in HEK 293T and HeLa CXCR4-KO had been substantially absent from the expression of CXCR4 protein as compared to the respective control cell lines. 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 adsorption and entry of EBOV VLPs into target cells, 600 μL of EBOV VLPs (10 5～106 copies/mL) were infected in the knockdown cell lines (HEK 293T CXCR4-KO and HeLa CXCR4-KO) and control cell lines (HEK 293T lenti-V2 and HELA LENTI-V2), respectively, and each cell line (CTRL) without EBOV VLPs was used as a control and either adsorbed at 4℃for 2h or placed at 37℃for 3h. Then extracting total RNA of each cell by TRIzol method, and obtaining cDNA by reverse transcription.
3. Real-time fluorescent quantitative PCR
The cDNA obtained in the above step was used as a template, and the level of GP mRNA adsorbed on target cells was detected by a real-time fluorescent quantitative PCR method using primers shown in Table 1, and the PCR reaction system and the PCR reaction conditions were the same as in example 3.
2. Experimental results
As shown in a and C in fig. 5, after EBOV VLPs 2h adsorption at 4 ℃, the levels of GP mRNA in HEK 293T cells and HeLa cells after CXCR4 knockout were significantly reduced compared to control cell lines.
As shown in B and D in fig. 5, after infection with EBOV VLPs for 3h at 37 ℃, the levels of GP mRNA in HEK 293T and HeLa cells after CXCR4 knockout were significantly reduced compared to the control cell line.
The results show that the CXCR4 gene in the knocked-out cell inhibits the adsorption of the EBOV VLPs and the target cell.
Example 5CXCR4 receptor antagonists inhibit the adsorption of EBOV VLPs and entry into target cells
1. Experimental method
1. Receptor antagonist treatment and cell infection
Pretreatment with CXCR4 receptor antagonist Motixafortide (HY-P0171, medChemExpress) at a final concentration of 20. Mu.M for 1h (with DMSO as a solvent control) followed by infection with 600. Mu.L of EBOV VLPs (10 5～106 copies/mL) was performed in HEK 293T cells and HeLa cells, respectively, and each cell line (CTRL) not infected with EBOV VLPs was subjected to adsorption at 4℃for 2h or at 37℃for 3h as a control. Then extracting total RNA of each cell by TRIzol method, and obtaining cDNA by reverse transcription.
2. Monoclonal antibody treatment and cell infection
HEK 293T cells were pretreated with CXCR 4-specific antibody 12G5 (16-9999-81, invitrogen) at a final concentration of 15. Mu.g/mL for 1h, heLa cells were pretreated with CXCR 4-specific antibody 12G5 at a final concentration of 20. Mu.g/mL for 1h, then 600. Mu.L EBOV VLPs (10 5～106 copies/mL) were infected, and each cell line (CTRL) without EBOV VLPs was used as a control and adsorbed at 4℃for 2h. Then extracting total RNA of each cell by TRIzol method, and obtaining cDNA by reverse transcription.
3. Real-time fluorescent quantitative PCR
The cDNA obtained in the above step was used as a template, and the level of GP mRNA adsorbed on target cells was detected by a real-time fluorescent quantitative PCR method using primers shown in Table 1, and the PCR reaction system and the PCR reaction conditions were the same as in example 3.
2. Experimental results
As shown in a and B in fig. 6, GP mRNA levels in HEK 293T cells and HeLa cells treated with CXCR4 receptor antagonist Motixafortide were significantly reduced after adsorption of EBOV VLPs for 2h at 4 ℃. CXCR4 receptor antagonists were shown to inhibit the adsorption of EBOV VLPs on the surface of target cells.
As shown in C and D in fig. 6, GP mRNA levels in HEK 293T cells and HeLa cells treated with CXCR4 receptor antagonist Motixafortide were significantly reduced after infection with EBOV VLPs for 3h at 37 ℃. CXCR4 receptor antagonists were shown to inhibit entry of EBOV VLPs into target cells.
As shown in E and F in fig. 6, after adsorption of EBOV VLPs for 2h at 4 ℃, GP mRNA levels were significantly reduced in HEK 293T and HeLa cells treated with CXCR4 monoclonal antibody 12G 5. Antibodies targeting the binding CXCR4 receptor were shown to block CXCR4 mediated adsorption and entry of EBOV VLPs into target cells.
It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and that other various changes and modifications can be made by one skilled in the art based on the above description and the idea, and it is not necessary or exhaustive to all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.
Claims (4)
- Use of an antagonist of cxcr4 receptor for the preparation of a product against ebola virus infection, characterized in that said antagonist is Motixafortide.
- 2. Use of an antibody to CXCR4 for the preparation of a product against ebola virus infection, wherein said antibody has clone number 12G5.
- 3. Use of a biological material for the preparation of a product resistant to ebola virus infection, characterized in that the biological material is any one of the following (1) - (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).
- 4. The use according to any one of claims 1 to 3, wherein said anti-ebola virus infection comprises inhibiting ebola virus adsorption and/or entry into a target cell.
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