Use of OAS2 gene and / or OAS2 protein in preparation of products against Ebola virus infection

Abstract

The application discloses application of OAS2 gene and / or OAS2 protein in preparation of products against Ebola virus infection. The application firstly confirms that OAS2 enhances the anti-Ebola virus ability of a host, inhibits expression of genes and proteins of Ebola virus, inhibits replication of a genome of Ebola virus, and has a significant inhibiting effect on various viruses of the Ebola virus genus. The application provides a theoretical basis for development of a new type of drug against Ebola virus infection, provides technical support for effective prevention of severe infectious diseases, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of virology, in particular, to application of OAS2 gene and / or OAS2 protein in preparation of products against Ebola virus infection. BACKGROUND

[0002] Ebola virus disease (EVD) is a severe and hemorrhagic infectious disease caused by infection of Ebola virus (EBOV) of Filoviridae. Although the Food and Drug Administration (FDA) approved monoclonal antibodies (mAb114, REGN-EB3, etc.) have shown certain anti-EBOV therapeutic effect, but also cannot make nearly one-third of patients die. So far, there is no effective EVD cure method. Therefore, it is essential to develop new and efficient anti-EBOV treatment means.

[0003] After the virus infects the host cell, the innate immune response of the body plays a key role as the first line of defense against viral infection. The 2-5A synthetase (OAS) family plays a key role in anti-viral infection, and its members include OAS 1, OAS2, OAS3 and OAS-like (OASL) proteins. Among them, OAS1, OAS2 and OAS3 have 2-5A synthetase activity, and can specifically synthesize 2-5A after binding with viral RNA. As a second messenger, 2-5A can bind to RNase L (RNase L), promote its dimerization and activate the endonuclease activity of RNase L, recognize UU / UA sites in viral RNA and intracellular RNA sequences, thereby degrading viral and intracellular RNA, and then inhibiting viral replication. However, recent studies have shown that the OAS / RNase L pathway in Zika virus (ZIKV) actually promotes the assembly of ZIKV gene factories, thereby promoting ZIKV replication. Therefore, for those skilled in the art, OAS / RNase L is likely to play different or even opposite roles in the process of host resistance to different RNA virus infections. So far, there is no report on the effect of OAS family or OAS / RNase L on host resistance to EBOV infection. SUMMARY

[0004] In order to solve the above problems existing in the prior art, the present application provides application of OAS2 gene and / or OAS2 protein in preparation of products against Ebola virus infection.

[0005] A first object of the present application is to provide an application of OAS2 gene and / or OAS2 protein in preparing a product against Ebola virus infection.

[0006] A second object of the present application is to provide an application of OAS2 gene and / or OAS2 protein in preparing a product for inhibiting gene and / or protein expression of Ebola virus.

[0007] A third object of the present application is to provide an application of OAS2 gene and / or OAS2 protein in preparing a product for inhibiting replication of Ebola virus.

[0008] A fourth object of the present application is to provide an application of biological material for promoting expression of OAS2 gene and / or OAS2 protein in preparing a product against Ebola virus infection.

[0009] A fifth object of the present application is to provide an application of biological material for promoting expression of OAS2 gene and / or OAS2 protein in preparing a product for inhibiting gene and / or protein expression of Ebola virus.

[0010] A sixth object of the present application is to provide an application of biological material for promoting expression of OAS2 gene and / or OAS2 protein in preparing a product for inhibiting replication of Ebola virus.

[0011] In order to achieve the above objects, the present application is achieved by the following solutions:

[0012] The applicant found in the previous research that proton pump inhibitor tenatoprazole can inhibit replication of Ebola virus genome, but the inhibition mechanism is still unclear. The present application found through RNA sequencing that OAS2 in OAS family is a key gene against Ebola virus. In addition, it was further found that overexpression of OAS2 can inhibit expression level of genes and proteins of Ebola virus and replication of genome, while the inhibition effect is reversed after knocking out OAS2 by CRISPR / cas9 gene knockout technology.

[0013] The present application claims the following:

[0014] An application of OAS2 gene and / or OAS2 protein in preparing a product against Ebola virus infection.

[0015] Preferably, the amino acid sequence of the OAS2 protein is shown as SEQ ID NO. 9.

[0016] Preferably, the nucleotide sequence of the nucleic acid molecule encoding the OAS2 protein is shown as SEQ ID NO. 32 or a complete complementary sequence of the sequence shown as SEQ ID NO. 32.

[0017] Preferably, the Ebola virus comprises any one or several of Zaire Ebola virus, Sudan Ebola virus, Tai Forest Ebola virus, Bundibugyo Ebola virus or Reston Ebola virus.

[0018] Use of OAS2 gene and / or OAS2 protein in the preparation of a product for inhibiting the expression of a gene and / or a protein of Ebola virus.

[0019] Preferably, the amino acid sequence of the OAS2 protein is as shown in SEQ ID NO. 9.

[0020] Preferably, the nucleotide sequence of the nucleic acid molecule encoding the OAS2 protein is as shown in SEQ ID NO. 32 or as shown in the full complement of the sequence shown in SEQ ID NO. 32.

[0021] Preferably, the Ebola virus comprises any one or several of Zaire Ebola virus, Sudan Ebola virus, Tai Forest Ebola virus, Bundibugyo Ebola virus or Reston Ebola virus.

[0022] Use of OAS2 gene and / or OAS2 protein in the preparation of a product for inhibiting the replication of Ebola virus.

[0023] Preferably, the amino acid sequence of the OAS2 protein is as shown in SEQ ID NO. 9.

[0024] Preferably, the nucleotide sequence of the nucleic acid molecule encoding the OAS2 protein is as shown in SEQ ID NO. 32 or as shown in the full complement of the sequence shown in SEQ ID NO. 32.

[0025] Preferably, the Ebola virus comprises any one or several of Zaire Ebola virus, Sudan Ebola virus, Tai Forest Ebola virus, Bundibugyo Ebola virus or Reston Ebola virus.

[0026] Use of a biological material for promoting the expression of OAS2 gene and / or OAS2 protein in the preparation of a product for resisting Ebola virus infection.

[0027] Preferably, the amino acid sequence of the OAS2 protein is as shown in SEQ ID NO. 9.

[0028] Preferably, the nucleotide sequence of the nucleic acid molecule encoding the OAS2 protein is as shown in SEQ ID NO. 32 or as shown in the full complement of the sequence shown in SEQ ID NO. 32.

[0029] Preferably, the biological material for promoting the expression of OAS2 gene and / or OAS2 protein comprises any one of the following (1) to (3):

[0030] (1) an expression cassette containing a nucleic acid molecule encoding the OAS2 protein;

[0031] (2) a recombinant expression vector containing the expression cassette in (1);

[0032] (3) a microorganism containing the recombinant expression vector in (2).

[0033] More preferably, the recombinant expression vector in (2) is a pCAGGS vector as a backbone vector.

[0034] Preferably, the Ebola virus includes any one or several of Zaire Ebola virus, Sudan Ebola virus, Tai Forest Ebola virus, Bundibugyo Ebola virus or Reston Ebola virus.

[0035] The biological material for promoting expression of the OAS2 gene and / or OAS2 protein is used in the preparation of a product for inhibiting expression of a gene and / or protein of Ebola virus.

[0036] Preferably, the amino acid sequence of the OAS2 protein is shown in SEQ ID NO. 9.

[0037] Preferably, the nucleotide sequence of the nucleic acid molecule encoding the OAS2 protein is shown in SEQ ID NO. 32 or a fully complementary sequence of the sequence shown in SEQ ID NO. 32.

[0038] Preferably, the biological material for promoting expression of the OAS2 gene and / or OAS2 protein includes any one of the following (1) to (3):

[0039] (1) an expression cassette containing a nucleic acid molecule encoding the OAS2 protein;

[0040] (2) a recombinant expression vector containing the expression cassette in (1);

[0041] (3) a microorganism containing the recombinant expression vector in (2).

[0042] More preferably, the recombinant expression vector in (2) is a pCAGGS vector as a backbone vector.

[0043] Preferably, the Ebola virus includes any one or several of Zaire Ebola virus, Sudan Ebola virus, Tai Forest Ebola virus, Bundibugyo Ebola virus or Reston Ebola virus.

[0044] The biological material for promoting expression of the OAS2 gene and / or OAS2 protein is used in the preparation of a product for inhibiting replication of Ebola virus.

[0045] Preferably, the amino acid sequence of the OAS2 protein is shown as SEQ ID NO. 9.

[0046] Preferably, the nucleotide sequence of the nucleic acid molecule encoding the OAS2 protein is shown as SEQ ID NO. 32 or a fully complementary sequence of the sequence shown as SEQ ID NO. 32.

[0047] Preferably, the biological material for promoting the expression of the OAS2 gene and / or OAS2 protein comprises any one of (1) to (3) below:

[0048] (1) an expression cassette containing a nucleic acid molecule encoding the OAS2 protein;

[0049] (2) a recombinant expression vector containing the expression cassette in (1);

[0050] (3) a microorganism containing the recombinant expression vector in (2).

[0051] More preferably, the recombinant expression vector in (2) uses a pCAGGS vector as a backbone vector.

[0052] Preferably, the Ebola virus includes any one or several of Zaire Ebola virus, Sudan Ebola virus, Tai Forest Ebola virus, Bundibugyo Ebola virus or Reston Ebola virus.

[0053] Compared with the prior art, the present application has the following beneficial effects:

[0054] The present application first explicitly determines that OAS2 enhances the anti-Ebola virus ability of the host, inhibits the expression of the genes and proteins of the Ebola virus, inhibits the replication of the genome of the Ebola virus, and has a significant inhibitory effect on various types of viruses of the genus Ebola. The present application provides a theoretical basis for developing new drugs against Ebola virus infection, provides technical support for effectively preventing severe infectious diseases, and has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0055] Figure 1 Determination of key genes against EBOV based on RNA sequencing; A is the differential expression gene analysis result of the uninfected group (CTRL), the untreated group (P1 EBOV) and the infected and treated group (P1 EBOV+tenatoprazole); B and C are the results of GO function enrichment analysis of DEGs of group 1 (CTRL vs. P1 EBOV) according to biological process and molecular function; D and E are the results of KEGG signal pathway enrichment analysis and GO function enrichment analysis of DEGs of group 2 (P1 EBOV vs. P1 EBOV+tenatoprazole); F is the DEGs Wayne analysis chart of group 1 and group 2.

[0056] Figure 2 Effect of Tegoprazan on mRNA expression level of OAS family; A-C are the detection results of mRNA expression level of OAS2, OAS1 and OAS3, respectively; *P<0.05, ***P<0.001, NS, non-significant, two-tailed t-test.

[0057] Figure 3 Effect of overexpression of OAS2 on mRNA expression level of each gene of EBOV; A-F are the detection results of mRNA expression level of GP, NP, VP40, VP35, VP30 and VP24 of EBOV, respectively; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, two-tailed t-test.

[0058] Figure 4 Effect of overexpression of OAS2 on expression level of each protein of EBOV; A-F are the detection results of protein expression level of EBOV GP, EBOV VP40, EBOV VP35, EBOV VP30, EBOV VP24 and EBOV NP, respectively; G is the detection result of protein expression level of EBOV GP under different doses of OAS2; H is the detection result of protein expression level of GP of different species of Ebola virus under overexpression of OAS2.

[0059] Figure 5 Effect of knockout of OAS2 gene on gene expression and genome replication of EBOV; A is the identification of knockout effect of OAS2 gene; B is the detection result of mRNA level of EBOV GP; C is the detection result of genome replication level of P1 generation of EBOV; ****P<0.0001, two-tailed t-test. DETAILED DESCRIPTION

[0060] The present application will be further described below in conjunction with the accompanying drawings and specific embodiments, which are only used to explain the present application and are not used to limit the scope of the present application. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

[0061] Example 1 Determination of key genes for anti-EBOV effect based on RNA sequencing

[0062] 1. Preparation of sequencing sample

[0063] The virus-like particles (trVLPs) system simulating the complete replication cycle of EBOV was established by using the existing technology (DOI: 10.3791 / 52381), and the P1 generation of EBOV virus-like particles (denoted as EBOV VLPs) was synthesized by using HEK 293T cells.

[0064] The HEK 293T cells cultured to the logarithmic growth phase were divided into an uninfected group (denoted as CTRL), an untreated group (denoted as P1 EBOV), and an infected treatment group (denoted as P1 EBOV+tenatoprazole). Among them, the HEK 293T cells in the uninfected group were directly cultured for 48 h; the HEK 293T cells in the untreated group were infected with 1×10 6 copies / mL of P1 generation of EBOV VLPs for 48 h; and the HEK 293T cells in the infected treatment group were infected with 1×10 6 copies / mL of P1 generation of EBOV and added with 10 μM tenatoprazole (manufacturer: MedChemExpress, catalog number: HY-17421) for treatment for 48 h. The total nucleic acid of the cells in the three groups was collected by using the TRIzol method as a sequencing sample.

[0065] 2. RNA sequencing and data analysis

[0066] The sequencing sample was sent to a company for RNA sequencing. According to the results of RNA sequencing, the genes with a horizontal change fold number of more than 2 were defined as differentially expressed genes (DEGs), and the gene expression change of the three groups was analyzed.

[0067] As shown in A of FIG. 1, Figure 1 Compared with the uninfected group, there were a total of 169 DEGs (denoted as Group 1) in the cells of the untreated group after P1 generation of EBOV infection, 99 genes were up-regulated, and 70 genes were down-regulated; compared with the untreated group, after P1 EBOV infection and tenatoprazole treatment, there were a total of 188 DEGs (denoted as Group 2) in the cells of the infected treatment group, 111 genes were up-regulated, and 77 genes were down-regulated.

[0068] The GO function enrichment analysis of the 169 DEGs in Group 1 was performed, as shown in FIG. 2, Figure 1As shown in B and C, these DEGs are enriched in innate immune-related pathways and molecular entries such as type I IFN pathway, viral response, and 2'-5' oligoadenylate synthase activity, indicating that EBOV infection successfully activates the host cell's innate immune response pathways. KEGG signaling pathway enrichment analysis and GO function enrichment analysis were performed on 188 DEGs in group 2, as shown in... Figure 1 As shown in D and E, these DEGs are enriched in innate immune pathways and molecular entries such as the TNF signaling pathway, JAK-STAT signaling pathway, and 2'-5' oligoadenylate synthase activity. These results suggest that tretinoin may exert its anti-EBOV effect by further activating the host's innate immune pathways.

[0069] Further identification of key genes for EBOV resistance and construction of Venn diagrams for groups 1 and 2, such as... Figure 1 As shown in F, the two groups of DEGs share 13 common DEGs, with OAS2 being one of them, while OAS1 and OAS3 are not among them. Furthermore, OAS2 is present in multiple entries in the GO function enrichment analysis of group 2.

[0070] 3. Effects of tretinoin on OAS family expression

[0071] Total nucleic acids from the uninfected, untreated, and infected groups were reverse transcribed to obtain cDNA, which was used as a template to detect the mRNA expression levels of OAS1, OAS2, and OAS3 by RT-qPCR. The primers used are shown in Table 1. The RT-qPCR reaction system consisted of: 10 μL 2×qPCR buffer, 1 μL F primer (final concentration 10 μM), 1 μL R primer (final concentration 10 μM), 7 μL H2O, and 1 μL cDNA. The RT-qPCR reaction program was: 95℃ for 10 min, 1 cycle; 95℃ for 15 s, 60℃ for 1 min, 40 cycles; melting curve: 95℃ for 15 s, 60℃ for 1 min, 95℃ for 1 s.

[0072] Table 1 Primers for RT-qPCR detection

[0073]

[0074]

[0075] like Figure 2 As shown in Figures A through C, P1 generation EBOV infection upregulated intracellular OAS1, OAS2, and OAS3 mRNA levels. Titaprazole treatment further upregulated OAS2 mRNA levels by nearly 5-fold, but did not further alter OAS1 and OAS3 mRNA levels. This indicates that titaprazole upregulated OAS2 expression.

[0076] The above results show that OAS2 gene is a key gene against Ebola virus.

[0077] Example 2 Effect of overexpression of OAS2 on gene and protein expression of Ebola virus and replication of genome

[0078] 1. Effect of overexpression of OAS2 on inhibition of gene and protein expression of EBOV

[0079] (1) Construction of recombinant expression vector

[0080] Referring to the method of "Construction of Fusion Protein Expression Plasmid" in the prior art (CN118252937A), an HA tag is fused to the carboxy terminus of the OAS2 protein (SEQ ID NO. 9), a 3xflag tag is fused to the carboxy terminus of the GP protein (SEQ ID NO. 10), the NP protein (SEQ ID NO. 11), the VP40 protein (SEQ ID NO. 12), and the VP30 protein (SEQ ID NO. 13) of the Zaire Ebola virus (EBOV), respectively, and a 3xmyc tag is fused to the carboxy terminus of the VP35 protein (SEQ ID NO. 14) and the VP24 protein (SEQ ID NO. 15), respectively. The coding sequences of the seven protein fragments are synthesized by gene synthesis, and are sequentially denoted as OAS2-HA, EBOV GP-3xflag, EBOV NP-3xflag, EBOV VP40-3xflag, EBOV VP35-3xflag, EBOV VP30-3xflag, and EBOV VP24-3xflag.

[0081] Subsequently, the recombinant expression vector of the OAS2 protein (denoted as pCAGGS-OAS2-HA), the recombinant expression vector of the EBOV GP protein (denoted as pCAGGS-GP-3xflag), the recombinant expression vector of the EBOV NP protein (denoted as pCAGGS-NP-3xflag), the recombinant expression vector of the EBOV VP40 protein (denoted as pCAGGS-VP40-3xflag), the recombinant expression vector of the EBOV VP35 protein (denoted as pCAGGS-VP35-3xmyc), the recombinant expression vector of the EBOV VP30 protein (denoted as pCAGGS-VP30-3xflag), and the recombinant expression vector of the EBOV VP24 protein (denoted as pCAGGS-VP24-3xmyc) are respectively constructed by taking the pCAGGS vector as a backbone.

[0082] (2) Cell transfection

[0083] 2000 ng pCAGGS-OAS2-HA and 2000 ng pCAGGS-GP-3×flag were transfected into HEK293T cells, designated as co-transfection group 1; 2000 ng pCAGGS-OAS2-HA and 2000 ng pCAGGS-NP-3×flag were transfected into HEK293T cells, designated as co-transfection group 2; 2000 ng pCAGGS-OAS2-HA and 2000 ng pCAGGS-VP40-3×flag were transfected into HEK293T cells, designated as co-transfection group 3; 2000 ng pCAGGS-OAS2-HA and 2000 ng pCAGGS-VP35-3×myc were transfected into HEK293T cells, designated as co-transfection group 4; 2000 ng pCAGGS-OAS2-HA and 2000 ng pCAGGS-GP-3×flag were transfected into HEK293T cells, designated as co-transfection group 5; 2000 ng pCAGGS-OAS2-HA and 2000 ng pCAGGS-GP-3×flag were transfected into HEK293T cells, designated as co-transfection group 6; 2000 ng pCAGGS-OAS2-HA and 2000 ng pCAGGS-GP-3×flag were transfected into HEK293T cells, designated as co-transfection group 7; 2000 ng pCAGGS-OAS2-HA and 2000 ng pCAGGS-GP-3×flag were transfected into HEK293T cells, designated as co-transfection group 8; 2000 ng pCAGGS-OAS2-HA and 2000 ng pCAGGS-GP-3×fla pCAGGS-VP30-3×flag was transfected into HEK 293T cells, designated as co-transfection group 5; 2000 ng pCAGGS-OAS2-HA and 2000 ng pCAGGS-VP24-3×myc were transfected into HEK 293T cells, designated as co-transfection group 6. At the same dosage, pCAGGS vector was co-transfected into HEK 293T cells with pCAGGS-GP-3×flag, pCAGGS-NP-3×flag, pCAGGS-VP40-3×flag, pCAGGS-VP35-3×myc, pCAGGS-VP30-3×flag, and pCAGGS-VP24-3×myc, respectively, serving as controls for co-transfection groups 1–6.

[0084] (3) RT-qPCR detection of gene mRNA expression level

[0085] Forty-eight hours after cell transfection, total nucleic acid was collected from co-transfected groups 1-6 and the control group. cDNA was obtained through reverse transcription and used as a template. The mRNA expression levels of each viral gene were detected by RT-qPCR. The primers used are shown in Table 2. The RT-qPCR reaction system and procedure were the same as in Example 1.

[0086] Table 2 Primers for RT-qPCR detection

[0087]

[0088] like Figure 3 As shown in Figures A through F, compared to the control, the mRNA levels of EBOV GP, NP, VP40, VP35, VP30, and VP24 in co-transformation groups 1 through 6 were significantly downregulated after OAS2 overexpression. This indicates that OAS2 overexpression inhibits the expression of EBOV viral genes.

[0089] (4) Western blot detection of protein expression levels

[0090] After 48h of cell transfection, total proteins of co-transfection groups 1-6 and their controls were collected, and the expression levels of each viral protein were detected by western blot. Alpha-tubulin protein was used as an internal reference protein. The antibodies used were as follows: OAS2 antibody (manufacturer: abcam; item number: ab197655), GP, VP40, VP30, and NP proteins were detected using anti-Flag tag protein antibody (manufacturer: Sigma-Aldrich; item number: F9291), VP35 and VP24 were detected using anti-Myc tag protein antibody (manufacturer: MBL; item number: M192-3), and alpha-tubulin antibody (manufacturer: MBL; item number: PM054).

[0091] As shown in A-F in FIG. 1, compared with the control, the protein expression levels of EBOV GP, VP40, VP35, VP30, VP24, and NP in co-transfection groups 1-6 were significantly down-regulated after overexpression of OAS2. This indicates that overexpression of OAS2 inhibits the expression of each viral protein of EBOV. Figure 4

[0092] 2, Effect of different doses of OAS2 on EBOV protein expression

[0093] Taking EBOV GP protein as a representative, the specific inhibitory effect of OAS2 on EBOV protein expression was further clarified.

[0094] (1) Cell transfection

[0095] 2000 ng of pCAGGS-GP-3xflag was transfected into HEK 293T cells, and different doses (0 ng, 200 ng, 400 ng, 800 ng, and 1600 ng) of pCAGGS-OAS2-HA were co-transfected into HEK 293T cells.

[0096] (2) Western blot detection of EBOV GP protein expression levels

[0097] After 48h of transfection, total proteins of co-transfection groups 1-6 and their controls were collected, and the expression levels of each viral protein were detected by western blot. Alpha-tubulin protein was used as an internal reference protein. The antibodies used were as follows: OAS2 antibody (manufacturer: abcam; item number: ab197655), GP, VP40, VP30, and NP proteins were detected using anti-Flag tag protein antibody (manufacturer: Sigma-Aldrich; item number: F9291), VP35 and VP24 were detected using anti-Myc tag protein antibody (manufacturer: MBL; item number: M192-3), and alpha-tubulin antibody (manufacturer: MBL; item number: PM054).

[0098] As shown in G in FIG. 2, with the increase of OAS2 protein expression level, the level of EBOV GP protein in the cells gradually decreased, showing a significant dose-dependent effect. Figure 4

[0099] 3, Effect of overexpression of OAS2 on protein expression of different types of Ebola virus ​​

[0100] To further determine whether OAS2 inhibits the expression of different types of Ebola virus GP proteins, represented by GP protein.

[0101] (1) Construction of recombinant expression vector

[0102] According to the method of the present embodiment, 3xflag tag was fused to the carboxy-terminal of the GP protein of Sudan Ebola virus (SUDV) (SEQ ID NO. 28), the GP protein of Tai Forest Ebola virus (TAFV) (SEQ ID NO. 29), the GP protein of Bundibugyo Ebola virus (BDBV) (SEQ ID NO. 30) and the GP protein of Reston Ebola virus (RESTV) (SEQ ID NO. 31), respectively, and then pCAGGS vector was used as a backbone to construct the recombinant expression vector of SUDV GP protein (denoted as pCAGGS-SUDV GP-3xflag), the recombinant expression vector of TAFV GP protein (denoted as pCAGGS-TAFV GP-3xflag), the recombinant expression vector of BDBV GP protein (denoted as pCAGGS-BDBV GP-3xflag) and the recombinant expression vector of RESTV GP protein (denoted as pCAGGS-RESTV GP-3xflag), respectively.

[0103] (2) Cell transfection

[0104] 2000 ng of pCAGGS-OAS2-HA was co-transfected into HEK 293T cells with 2000 ng of pCAGGS-GP-3xflag, 2000 ng of pCAGGS-SUDV GP-3xflag, 2000 ng of pCAGGS-TAFV GP-3xflag, 2000 ng of pCAGGS-BDBV GP-3xflag and 2000 ng of pCAGGS-RESTV GP-3xflag, respectively; and pCAGGS vector was co-transfected into HEK 293T cells with pCAGGS-GP-3xflag, pCAGGS-SUDV GP-3xflag, pCAGGS-TAFV GP-3xflag, pCAGGS-BDBV GP-3xflag and pCAGGS-RESTV GP-3xflag, respectively, as a control.

[0105] (3) Western blot detection of the expression level of different types of Ebola virus GP protein

[0106] The expression levels of OAS2, EBOV GP protein, SUDV GP protein, TAFV GP protein, BDBV GP protein and RESTV GP protein in each cell were detected by western blot.

[0107] As shown by H in Figure 4 Compared with the control, the expression levels of the five Ebola virus GP proteins in the cells after overexpression of OAS2 were significantly decreased. This indicates that OAS2 has a broad anti-Ebola virus effect.

[0108] Example 3 Effect of knocking out OAS2 on the gene expression and genome replication of Ebola virus

[0109] 1. Construction of OAS2 gene knockout cells

[0110] To further clarify the anti-EBOV effect of OAS2, OAS2 gene knockout HEK 293T cells were constructed.

[0111] (1) Construction of gene editing plasmid

[0112] The gRNA was designed according to the CDS region of the OAS2 gene (SEQ ID NO. 32), and the F primer for synthesizing the gRNA was 5'-CACCGTGCACCAGCTCCAATCAGCG-3' (SEQ ID NO. 33), and the R primer was 5'-AAACCGCTGATTGGAGCTGGTGCAC-3' (SEQ ID NO. 34).

[0113] 5 μL F primer (SEQ ID NO. 33), 5 μL R primer (SEQ ID NO. 34), 2 μL T4 DNA ligase buffer and 8 μL water (total 20 μL) were boiled at 100°C for 10 min, and then naturally cooled to room temperature to obtain the annealing product. The lenti CRISPR-V2 vector was digested with BsmBI enzyme at 37°C for 2 h, and then linearized lenti CRISPR-V2 vector was obtained by agarose gel electrophoresis and gel recovery.

[0114] The annealing product and the linearized lenti CRISPR-V2 vector were connected by T4 ligase overnight to obtain a gene editing vector for knocking out the OAS2 gene, which was denoted as lenti-CRISPR-OAS2 vector.

[0115] (2) Lentivirus packaging and infection

[0116] The lenti-CRISPR-CXCR4 vector, the helper vector pVSVg vector and the psPAX2 vector were co-transfected into HEK 293T cells according to a mass ratio of 6:3:4.5, and polybrene was added to a final concentration of 10 μg / mL to promote infection. The medium was changed 24 h after transfection. After another 24 h, puromycin was added to obtain single cell clones, and western blot was performed to identify the single cell clones (the antibodies used are as follows: OAS2 antibody (manufacturer: abcam; item number: ab197655), and a-tubulin protein as the internal reference protein (manufacturer: MBL; item number: PM054), and the identification result is consistent with the expectation, that is, the OAS2 gene knockout HEK 293T cell is recorded as OAS2-KO cell. The wild type HEK 293T cell without OAS2 gene knockout is used as a control cell line, which is recorded as CTRL cell.

[0117] As shown in Figure 5 As shown in A of FIG. 6, compared with the control cell line, the protein level of OAS2 in the OAS2 gene knockout HEK 293T cell is significantly reduced. It is shown that the OAS2 gene knockout HEK 293T cell is successfully constructed.

[0118] 2, Effect of OAS2 knockout on the expression of EBOV genes

[0119] (1) Cell transfection

[0120] EBOV GP was taken as a representative to explore the effect of OAS2 gene knockout on the expression level of EBOV GP mRNA. pCAGGS-GP-3×flag was transfected into OAS2-KO cells and CTRL cells at 3000 ng respectively.

[0121] (2) RT-qPCR detection of gene mRNA expression level

[0122] After 48 h of cell transfection, the total nucleic acid of each cell was collected, and cDNA was obtained by reverse transcription, which was used as a template to detect the mRNA expression level of each viral gene by RT-qPCR. The primers for detecting GP and ACTB are shown in Table 2. The RT-qPCR reaction system and reaction program are the same as those in Example 1.

[0123] As shown in Figure 5 As shown in B of FIG. 7, compared with the CTRL cell, the GP mRNA level in the OAS2-KO cell after transfection of pCAGGS-GP-3×flag is significantly increased. It is shown that knockout of OAS2 promotes the expression of EBOV viral genes.

[0124] 3, Effect of OAS2 knockout on the replication of EBOV genome

[0125] P1 generation EBOV at 1×10 6 OAS2-KO cells and CTR L cells were infected with a dose of copies / mL, respectively. After 48 h of infection, the fluorescence level in each cell was detected by a dual-luciferase reporter assay to evaluate the effect of OAS2 gene knockout on P1 generation EBOV genome replication.

[0126] like Figure 5 As shown in Figure C, compared with CTRL cells, the replication level of P1 generation EBOV was significantly increased after infection of OAS2-KO cells. This indicates that knockout of OAS2 promotes EBOV genome replication.

[0127] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. Promoting OAS2 the use of a biomaterial that promotes the expression of a gene and / or an OAS2 protein in the manufacture of a product for combating an Ebola virus infection, characterized in that, The promoting OAS2 The biological material that promotes the expression of the gene and / or OAS2 protein includes any one of the following (1) to (3): (1) an expression cassette containing a nucleic acid molecule encoding the OAS2 protein; (2) a recombinant expression vector containing the expression cassette in (1); (3) a microorganism containing the recombinant expression vector in (2).

2. Use according to claim 1, characterized in that, The amino acid sequence of the OAS2 protein is shown as SEQ ID NO.

9.

3. Use according to claim 1, characterized in that, The nucleotide sequence of the nucleic acid molecule encoding the OAS2 protein is shown as SEQ ID NO. 32 or as the fully complementary sequence of the sequence shown as SEQ ID NO.

32.

4. Use according to any one of claims 1 to 3, characterized in that, The Ebola virus includes any one or several of Zaire Ebola virus, Sudan Ebola virus, Tai Forest Ebola virus, Bundibugyo Ebola virus or Reston Ebola virus.

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