Application of OAS2 gene and / or OAS2 protein in preparation of product for resisting Ebola virus infection

Abstract

The invention discloses an application of an OAS2 gene and / or an OAS2 protein in preparation of a product for resisting Ebola virus infection. According to the application disclosed by the invention, the fact that OAS2 enhances the Ebola virus resistance of a host, inhibits the expression of genes and proteins of the Ebola virus and inhibits genome replication of the Ebola virus is determined for the first time, and the OAS2 has a remarkable inhibiting effect on various types of viruses of the Ebola virus. The invention provides a theoretical basis for developing novel anti-Ebola virus infection medicines, provides technical support for effectively preventing fulminating infectious diseases, and has wide application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of virology, and specifically, to the application of OAS2 gene and / or OAS2 protein in the preparation of products for anti-Ebola virus infection. Background Art

[0002] Ebola virus disease (EVD) is a severe hemorrhagic infectious disease caused by the infection of Ebola virus (EBOV) of the Filoviridae family. Although the monoclonal antibodies (such as mAb114, REGN-EB3, etc.) approved by the Food and Drug Administration (FDA) of the United States have shown certain anti-EBOV therapeutic effects, they still cannot save nearly one-third of patients from death. So far, there is no effective cure for EVD. Therefore, it is crucial to develop new and efficient anti-EBOV treatment means.

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

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

[0005] The first object of the present invention is to provide the use of the OAS2 gene and / or OAS2 protein in the preparation of products against Ebola virus infection.

[0006] The second object of the present invention is to provide the use of the OAS2 gene and / or OAS2 protein in the preparation of products for inhibiting the gene and / or protein expression of Ebola virus.

[0007] The third object of the present invention is to provide the use of the OAS2 gene and / or OAS2 protein in the preparation of products for inhibiting the replication of Ebola virus.

[0008] The fourth object of the present invention is to provide the use of biomaterials that promote the expression of the OAS2 gene and / or OAS2 protein in the preparation of products against Ebola virus infection.

[0009] The fifth object of the present invention is to provide the use of biomaterials that promote the expression of the OAS2 gene and / or OAS2 protein in the preparation of products for inhibiting the gene and / or protein expression of Ebola virus.

[0010] The sixth object of the present invention is to provide the use of biomaterials that promote the expression of the OAS2 gene and / or OAS2 protein in the preparation of products for inhibiting the replication of Ebola virus.

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

[0012] In previous studies by the applicant, it was found that the proton pump inhibitor tenatoprazole can inhibit the replication of the Ebola virus genome, but its inhibitory mechanism is still unclear. Through RNA sequencing, the present invention found that OAS2 in the OAS family is a key gene against the Ebola virus. In addition, it was further found that overexpression of OAS2 inhibits the expression levels of the genes and proteins of the Ebola virus and genomic replication, while knocking out OAS2 by the CRISPR / cas9 gene knockout technology reverses this inhibitory effect.

[0013] The present invention claims protection for the following:

[0014] The use of the OAS2 gene and / or OAS2 protein in the preparation of products against Ebola virus infection.

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

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

[0017] Preferably, the Ebola virus includes any one or more of Zaire Ebola virus, Sudan Ebola virus, Taï Forest Ebola virus, Bundibugyo Ebola virus, or Reston Ebola virus.

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

[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 the complete complementary sequence of the sequence shown in SEQ ID NO.32.

[0021] Preferably, the Ebola virus includes any one or more of Zaire Ebola virus, Sudan Ebola virus, Taï Forest Ebola virus, Bundibugyo Ebola virus, or Reston Ebola virus.

[0022] Use of the OAS2 gene and / or OAS2 protein in the preparation of a product for inhibiting the replication of the 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 the complete complementary sequence of the sequence shown in SEQ ID NO.32.

[0025] Preferably, the Ebola virus includes any one or more of Zaire Ebola virus, Sudan Ebola virus, Taï Forest Ebola virus, Bundibugyo Ebola virus, or Reston Ebola virus.

[0026] Use of a biomaterial for promoting the expression of the OAS2 gene and / or OAS2 protein in the preparation of a product for preventing 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 the complete complementary sequence of the sequence shown in SEQ ID NO.32.

[0029] Preferably, the biomaterial for promoting the expression of the OAS2 gene and / or OAS2 protein includes 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 described in (1);

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

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

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

[0035] Use of a biological material that promotes the expression of the OAS2 gene and / or OAS2 protein in the preparation of a product for inhibiting the expression of the Ebola virus gene and / or protein.

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

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

[0038] Preferably, the biological material that promotes the 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 described in (1);

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

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

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

[0044] Use of a biological material that promotes the expression of the OAS2 gene and / or OAS2 protein in the preparation of a product for inhibiting the replication of the Ebola virus.

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

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

[0047] Preferably, the biomaterial for promoting the expression of the OAS2 gene and / or the OAS2 protein includes any one of the following (1) to (3):

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

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

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

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

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

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

[0054] The present invention has for the first time clearly demonstrated that OAS2 enhances the host's anti-Ebola virus ability, inhibits the expression of the genes and proteins of the Ebola virus, inhibits the genomic replication of the Ebola virus, and has a significant inhibitory effect on various types of viruses in the genus Ebola. The present invention provides a theoretical basis for the development of new drugs against Ebola virus infection, provides technical support for the effective prevention and control of highly infectious diseases, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 To determine the key genes against EBOV based on RNA sequencing; A shows the results of differential expression gene analysis of the uninfected group (CTRL), the untreated group (P1EBOV), and the infected and treated group (P1 EBOV+tenatoprazole); B and C show the results of GO functional enrichment analysis of the DEGs of group 1 (CTRL vs. P1 EBOV) according to biological process and molecular function in sequence; D and E show the results of KEGG signaling pathway enrichment analysis and GO functional enrichment analysis of the DEGs of group 2 (P1 EBOV vs. P1 EBOV+tenatoprazole) in sequence; F shows the Venn analysis diagram of the DEGs of group 1 and group 2.

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

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

[0058] Figure 4 Effect of overexpressing OAS2 on the protein expression levels of each protein of EBOV; the detection results of the protein expression levels of EBOV GP, EBOV VP40, EBOV VP35, EBOV VP30, EBOV VP24, and EBOV NP are shown in A - F in sequence; G shows the detection results of the EBOV GP protein expression level under different doses of OAS2; H shows the detection results of the GP protein expression levels of different Ebola virus species under overexpressed OAS2.

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

[0060] The present invention will be further elaborated in detail below in combination with the accompanying drawings of the specification and specific embodiments. The embodiments 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 embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used are all reagents and materials that can be obtained from commercial channels unless otherwise specified.

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

[0062] 1. Preparation of sequencing samples

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

[0064] 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 and treated 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 the P1 generation of EBOV VLPs for 48 h; the HEK 293T cells in the infected and treated group were infected with 1×10 6 copies / mL of the P1 generation of EBOV and 10 μM tenatoprazole (manufacturer: MedChemExpress, catalog number: HY-17421) was added, and the treatment was carried out for 48 h. The total nucleic acids of the three groups of cells were collected by the TRIzol method and used as sequencing samples.

[0065] 2. RNA Sequencing and Data Analysis

[0066] The sequencing samples were sent to the company for RNA sequencing. According to the results of RNA sequencing, genes with a fold change in expression level exceeding 2-fold were defined as differentially expressed genes (DEGs), and the gene expression changes in the three groups were analyzed.

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

[0068] GO functional enrichment analysis was performed on the 169 DEGs in Group 1, as Figure 1As shown in B and C, these DEGs were enriched in innate immune-related pathways and molecular terms such as type I IFN pathway, viral response, 2'-5'-oligoadenylate synthetase activity, indicating that EBOV infection successfully activated the innate immune response pathway of host cells. KEGG pathway enrichment analysis and GO function enrichment analysis were performed on 188 DEGs in group 2. As Figure 1 shown in D and E, these DEGs were enriched in innate immune pathways and molecular terms such as TNF signaling pathway, JAK-STAT signaling pathway, and 2'-5'-oligoadenylate synthetase activity. The above results suggest that taitozole may play an anti-EBOV role by further activating the host innate immune pathway.

[0069] To further identify the key genes against EBOV, a Venn diagram of group 1 and group 2 was constructed. As Figure 1 shown in F, there were 13 common DEGs between the two groups of DEGs, and OAS2 was one of them, while OAS1 and OAS3 were not. And OAS2 was present in multiple terms in the GO function enrichment analysis of group 2.

[0070] 3. Effects of taitozole on the expression of OAS family

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

[0072] Table 1 RT-qPCR detection primers

[0073]

[0074]

[0075] As Figure 2 shown in A - C, P1 generation EBOV infection upregulated the mRNA levels of OAS1, OAS2, and OAS3 in cells, and under the treatment of taitozole, the OAS2 mRNA level was further upregulated nearly 5-fold, but did not further change the OAS1 and OAS3 mRNA levels. It shows that taitozole upregulates the expression of OAS2.

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

[0077] Example 2 Effects of overexpressing OAS2 on gene and protein expression of Ebola virus and replication of its genome

[0078] 1. Effects of overexpressing OAS2 on inhibiting gene and protein expression of EBOV

[0079] (1) Construction of recombinant expression vectors

[0080] Referring to the method of "construction of fusion protein expression plasmid" in the prior art (CN118252937A), an HA tag was fused to the carboxyl terminus of the OAS2 protein (SEQ ID NO.9), and 3×flag tags were respectively fused to the carboxyl termini of the GP protein (SEQ ID NO.10), NP protein (SEQ ID NO.11), VP40 protein (SEQ ID NO.12), and VP30 protein (SEQ ID NO.13) of Ebola virus (EBOV) of the Zaire strain. 3×myc tags were respectively fused to the carboxyl termini of the VP35 protein (SEQ ID NO.14) and VP24 protein (SEQ ID NO.15); the coding sequences of 7 protein fragments were synthesized by gene synthesis and were sequentially denoted as OAS2-HA, EBOV GP-3×flag, EBOV NP-3×flag, EBOV VP40-3×flag, EBOV VP35-3×flag, EBOV VP30-3×flag, and EBOV VP24-3×myc.

[0081] After that, using the pCAGGS vector as the backbone, recombinant expression vectors of the OAS2 protein (denoted as pCAGGS-OAS2-HA), EBOV GP protein (denoted as pCAGGS-GP-3×flag), EBOV NP protein (denoted as pCAGGS-NP-3×flag), EBOV VP40 protein (denoted as pCAGGS-VP40-3×flag), EBOV VP35 protein (denoted as pCAGGS-VP35-3×myc), EBOV VP30 protein (denoted as pCAGGS-VP30-3×flag), and EBOV VP24 protein (denoted as pCAGGS-VP24-3×myc) were respectively constructed.

[0082] (2) Cell transfection

[0083] 2000 ng of pCAGGS - OAS2 - HA and 2000 ng of pCAGGS - GP - 3×flag were transfected into HEK 293T cells, denoted as co - transfection group 1; 2000 ng of pCAGGS - OAS2 - HA and 2000 ng of pCAGGS - NP - 3×flag were transfected into HEK293T cells, denoted as co - transfection group 2; 2000 ng of pCAGGS - OAS2 - HA and 2000 ng of pCAGGS - VP40 - 3×flag were transfected into HEK 293T cells, denoted as co - transfection group 3; 2000 ng of pCAGGS - OAS2 - HA and 2000 ng of pCAGGS - VP35 - 3×myc were transfected into HEK 293T cells, denoted as co - transfection group 4; 2000 ng of pCAGGS - OAS2 - HA and 2000 ng of pCAGGS - VP30 - 3×flag were transfected into HEK 293T cells, denoted as co - transfection group 5; 2000 ng of pCAGGS - OAS2 - HA and 2000 ng of pCAGGS - VP24 - 3×myc were transfected into HEK 293T cells, denoted as co - transfection group 6. According to the same dose, the pCAGGS vector was co - transfected 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 into HEK 293T cells respectively, serving as the controls for co - transfection groups 1 - 6 in turn.

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

[0085] 48 h after cell transfection, the total nucleic acids of cells in co - transfection groups 1 - 6 and their controls were collected, reverse - transcribed to obtain cDNA, used as templates, and the mRNA expression levels of each viral gene were detected by RT - qPCR. The detection primers used are shown in Table 2. The RT - qPCR reaction system and reaction program were the same as those in Example 1.

[0086] Table 2 RT - qPCR detection primers

[0087]

[0088] As Figure 3 shown in A - F below, compared with the controls, the mRNA levels of EBOV GP, NP, VP40, VP35, VP30 and VP24 in co - transfection groups 1 - 6 were significantly down - regulated after over - expression of OAS2. It indicates that over - expression of OAS2 inhibits the expression of EBOV viral genes.

[0089] (4) Detecting protein expression levels by Western blot

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

[0091] As Figure 4 shown in A - F of

[0092] 2. Effects of different doses of OAS2 on EBOV protein expression

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

[0094] (1) Cell transfection

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

[0096] (2) Detecting the expression level of EBOV GP protein by Western blot

[0097] At 48 h after transfection, total proteins of the cells were collected, and the expression levels of OAS2 and EBOV GP proteins were detected by Western blot.

[0098] As Figure 4 shown in G of

[0099] 3. Effects of overexpressing OAS2 on the protein expression of different Ebola virus species

[0100] Taking the GP protein as an example, further clarify whether OAS2 has an inhibitory effect on the expression of GP proteins of different Ebola virus species.

[0101] (1) Construction of recombinant expression vectors

[0102] According to the method of this example, a 3×flag tag was fused to the carboxyl terminus 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. Then, using the pCAGGS vector as the backbone, recombinant expression vectors of SUDV GP protein (denoted as pCAGGS-SUDV GP-3×flag), TAFV GP protein (denoted as pCAGGS-TAFV GP-3×flag), BDBV GP protein (denoted as pCAGGS-BDBV GP-3×flag), and RESTV GP protein (denoted as pCAGGS-RESTV GP-3×flag) were constructed respectively.

[0103] (2) Cell transfection

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

[0105] (3) Detection of the expression levels of GP proteins of different Ebola virus species by western blot

[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 Figure 4 shown by H in , compared with the control, the expression levels of the 5 Ebola virus GP proteins in the cells decreased significantly after overexpression of OAS2. This indicates that OAS2 has a broad anti-Ebola virus effect.

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

[0109] 1. Construction of OAS2 gene knockout cells

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

[0111] (1) Construction of gene editing plasmid

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

[0113] 5 μL of the F primer (SEQ ID NO.33), 5 μL of the R primer (SEQ ID NO.34), 2 μL of T4 DNA ligase buffer and 8 μL of water (a total of 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 and placed at 37 °C for 2 h of digestion, and then linearized lenti CRISPR-V2 vector was obtained through agarose gel electrophoresis and gel recovery.

[0114] The annealing product was ligated overnight with the linearized lenti CRISPR-V2 vector through T4 ligase to obtain a gene editing vector for knocking out the OAS2 gene, denoted as the lenti-CRISPR-OAS2 vector.

[0115] (2) Lentivirus packaging and infection

[0116] The lenti - CRISPR - CXCR4 vector was co - transfected with the helper vectors pVSVg vector and psPAX2 vector into HEK 293T cells at a mass ratio of 6:3:4.5, and polybrene with a final concentration of 10 μg / mL was added to promote infection. The medium was changed 24 h after transfection. After another 24 h, puromycin was added to screen for single - cell clones, and the single - cell clones were identified by western blot (the antibodies used were as follows: OAS2 antibody (manufacturer: abcam; catalog number: ab197655), α - tubulin protein as the internal reference protein (manufacturer: MBL; catalog number: PM054)). Those with identification results meeting the expectations were HEK 293T cells with OAS2 gene knockout, denoted as OAS2 - KO cells. Wild - type HEK 293T cells without OAS2 gene knockout were used as the control cell line, denoted as CTRL cells.

[0117] As Figure 5 shown in A, compared with the control cell line, the protein level of OAS2 in HEK 293T cells with OAS2 gene knockout decreased significantly. This indicates that the construction of HEK 293T cells with OAS2 gene knockout was successful.

[0118] 2. Effects of OAS2 knockout on EBOV gene expression

[0119] (1) Cell transfection

[0120] Taking EBOV GP as an example, the effect of OAS2 gene knockout on the mRNA expression level of EBOV GP was explored. 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] 48 h after cell transfection, the total nucleic acids of each cell were collected, reverse - transcribed into cDNA, and used as templates to detect the mRNA expression levels 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 were the same as in Example 1.

[0123] As Figure 5 shown in B, compared with CTRL cells, the GP mRNA level in OAS2 - KO cells transfected with pCAGGS - GP - 3×flag increased significantly. This indicates that knocking out OAS2 promotes the expression of EBOV viral genes.

[0124] 3. Effects of OAS2 knockout on EBOV genome replication

[0125] The P1 generation of EBOV was used to infect OAS2-KO cells and CTRL cells respectively at a dose of 1×10 6 copies / mL. After 48 hours of infection, the fluorescence levels in each cell were detected by the dual-luciferase reporter assay to evaluate the effect of knocking out the OAS2 gene on the genomic replication of the P1 generation of EBOV.

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

[0127] 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 those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description and ideas. It is not necessary and impossible to list all the implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. Use of the OAS2 gene and / or OAS2 protein in the preparation of a product for anti-Ebola virus infection.

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

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

4. Use of a biomaterial that promotes the expression of the OAS2 gene and / or OAS2 protein in the preparation of a product for anti-Ebola virus infection.

5. Use of a biomaterial that promotes the expression of the OAS2 gene and / or OAS2 protein in the preparation of a product for inhibiting the gene and / or protein expression of Ebola virus.

6. Use of a biomaterial that promotes the expression of the OAS2 gene and / or OAS2 protein in the preparation of a product for inhibiting the replication of Ebola virus.

7. The application according to any one of claims 1 to 6, characterized in that, The amino acid sequence of the OAS2 protein is as shown in SEQ ID NO.

9.

8. The application according to any one of claims 1 to 6, characterized in that, The nucleotide sequence of the nucleic acid molecule encoding the OAS2 protein is as shown in SEQ ID NO.32 or the complete complementary sequence of the sequence shown in SEQ ID NO.

32.

9. The application according to any one of claims 4 to 6, characterized in that The biomaterial that promotes the expression of the OAS2 gene and / or OAS2 protein includes any one of the following (1) to (3): (1) An expression cassette containing the nucleic acid molecule encoding the OAS2 protein; (2) A recombinant expression vector containing the expression cassette described in (1); (3) A microorganism containing the recombinant expression vector described in (2).

10. The application according to any one of claims 1 to 9, characterized in that, The Ebola virus includes any one or more of Ebola virus Zaire, Ebola virus Sudan, Ebola virus Taï Forest, Ebola virus Bundibugyo, or Ebola virus Reston.

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