Application of SLFN12 protein in preparation of medicine for resisting small RNA viridae viruses

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of SLFN12 protein in preparation of a medicine for resisting small RNA viridae viruses. The invention accidentally discovers that the replication levels of EV71, EMCV, SVA and FMDV can be reduced by adding the SLFN12 protein into a culture medium for culturing EV71, EMCV, SVA and FMDV, which indicates that the SLFN12 protein has the effect of inhibiting the replication of small RNA viridae viruses, can be used for preparing drugs or adjuvants for resisting virus infection, and is used for inhibiting the replication of small RNA viridae viruses; secondly, the cell line after interference of SLFN12 protein expression in host cells can significantly promote replication of small RNA viridae viruses, can be used as a production cell line of small RNA viridae viruses or viral vaccines, and is used for expression production of small RNA viridae viruses or viral vaccines.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to the application of SLFN12 protein in the preparation of drugs against Picornaviridae viruses. Background Art

[0002] Picornaviridae is a family composed of the smallest group of RNA viruses. The viruses of this family are a class of single-stranded positive-strand RNA viruses widely present in humans and animals, which can cause a variety of serious infectious diseases, posing a serious threat to human health and animal husbandry. Representative viruses include Enterovirus 71 (EV71) in the genus Enterovirus, Encephalomyocarditis virus (EMCV) in the genus Cardiovirus, Seneca virus (SVA) in the genus Seneca virus, and Foot-and-mouth disease virus (FMDV) in the genus Aphthovirus, etc. EV71 is a picornavirus that mainly infects children and can cause a variety of diseases; EMCV is a picornavirus that is pathogenic to a variety of animals and humans; both have high pathogenicity, especially causing great harm in terms of nervous system and myocardial damage. Foot-and-mouth disease is a highly contagious and acute febrile animal infectious disease caused by foot-and-mouth disease virus, mainly infecting cloven-hoofed animals (such as pigs, cattle, sheep, etc.), posing a serious threat to the global livestock industry and being difficult to prevent for a long time; SVA is a pathogen that has attracted attention in pig herds in recent years and can cause swine vesicular disease; both FMDV and SVA are pathogens that cause great harm to the livestock industry, with similar clinical symptoms and causing great harm to the pig industry, making the prevention and control situation of this disease extremely severe.

[0003] SLFN12 protein is an important member of the Schlafen family of proteins. Members of this family are related to a variety of functions, such as inhibiting cell proliferation and promoting cell differentiation, inhibiting the migration and invasion of cancer cells, and enhancing sensitivity to DNA damage drugs. SLFN12 is expressed in monocytes, monocyte-derived dendritic cells, and T cells. In recent years, SLFN12 protein has attracted extensive attention in the fields of tumor and immunotherapy. The potential role of SLFN12 in the tumor microenvironment. SLFN12 can promote the proliferation, migration, and macrophage recruitment of glioma cells, thus affecting tumor progression and immunotherapy effects. Through single-cell RNA sequencing and transcriptome data analysis, SLFN12 was identified as an independent biomarker for predicting anti-PD-1 immunotherapy response, suggesting its potential application value in tumor immunotherapy. However, the role of SLFN12 protein in viral diseases has not been studied. Summary of the Invention

[0004] In view of the above technical problems, the present invention provides the application of SLFN12 protein in the preparation of drugs against Picornaviridae viruses. Specifically, it includes the following content:

[0005] In a first aspect, the present invention provides the use of an SLFN12 protein or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing or treating picornavirus infection.

[0006] Preferably, the picornavirus is selected from EV71, EMCV, SVA, FMDV.

[0007] Preferably, the SLFN12 protein or a pharmaceutically acceptable salt thereof is combined with a pharmaceutically acceptable carrier and / or excipient to form any pharmaceutically acceptable dosage form.

[0008] Preferably, the dosage form includes freeze-dried powder for injection, capsules, tablets, suspensions.

[0009] In a second aspect, the present invention provides the use of an SLFN12 protein or a pharmaceutically acceptable salt thereof in the preparation of an adjuvant for a picornavirus vaccine.

[0010] Preferably, the picornavirus is selected from EV71, EMCV, SVA, FMDV.

[0011] Preferably, the SLFN12 protein or a pharmaceutically acceptable salt thereof is combined with a pharmaceutically acceptable carrier and / or excipient to form any pharmaceutically acceptable dosage form.

[0012] Preferably, the dosage form includes freeze-dried powder for injection, capsules, tablets, suspensions.

[0013] In a third aspect, the present invention provides the use of an SLFN12 protein knockout / knockdown cell line as a production cell line for picornavirus or virus vaccine.

[0014] Preferably, the picornavirus is selected from EV71, EMCV, SVA, FMDV.

[0015] In a fourth aspect, the present invention provides the use of a reagent for interfering with / inhibiting the expression of SLFN12 protein in the preparation of a production synergist for picornavirus or virus vaccine.

[0016] Preferably, the picornavirus is selected from EV71, EMCV, SVA, FMDV.

[0017] In a fifth aspect, the present invention provides an siRNA for interfering with the expression of SLFN12 protein, and the sequence of the siRNA is:

[0018] F: 5’-GAAAGUGUCUCACGAGCUA(dT)(dT)-3’;

[0019] R: 5’-UAGCUCGUGAGACACUUUC(dT)(dT)-3’。

[0020] In a sixth aspect, the present invention provides the use of the siRNA described in the fifth aspect above in the preparation of a production cell line for picornavirus or virus vaccine.

[0021] Preferably, the picornavirus is selected from EV71, EMCV, SVA, FMDV.

[0022] The beneficial effects of the present invention are as follows: The present invention unexpectedly discovers that adding SLFN12 protein to the culture medium for culturing EV71, EMCV, SVA, and FMDV can reduce the replication levels of EV71, EMCV, SVA, and FMDV, indicating that the SLFN12 protein has the effect of inhibiting the replication of picornaviruses and can be used to prepare antiviral drugs or adjuvants for inhibiting the replication of picornaviruses; secondly, the cell line obtained by interfering with the expression of SLFN12 protein in host cells can significantly promote the replication of picornaviruses and can be used as a production cell line for picornaviruses or virus vaccines for the expression and production of picornaviruses or virus vaccines. Description of the Drawings

[0023] Figure 1 Detection results of viral mRNA content after overexpressing Flag-SLFN12 in HEK-293 cells for 24 hours and then infecting with EV71, EMCV, and SVA;

[0024] Figure 2 Detection results of 3D protein after overexpressing Flag-SLFN12 in HEK-293 cells for 24 hours and then infecting with EV71 and detection results of VP2 protein after infecting with SVA;

[0025] Figure 3 Detection results of SLFN12 after interfering with SLFN12 in HELA cells for 36 hours and then infecting with SVA;

[0026] Figure 4 Detection results of viral mRNA content and VP2 protein after interfering with SLFN12 in HELA cells for 36 hours and then infecting with SVA;

[0027] Figure 5 Detection results of viral mRNA content and virus titer after overexpressing Flag-SLFN12 in HEK-293 cells for 12 hours and then infecting with SVA;

[0028] Figure 6 Detection results of viral fluorescence after overexpressing Flag-SLFN12 in HEK-293 cells for 12 hours and then infecting with SVA;

[0029] Figure 7 Detection results of viral mRNA content after overexpressing SLFN12 in PK-15 cells for 24 hours and then infecting with FMDV;

[0030] Figure 8 Detection results of viral titer after overexpressing SLFN12 in PK-15 cells for 24 hours and then infecting with FMDV. Detailed implementation manners

[0031] Unless otherwise specified, the experimental methods in the following examples are all conventional methods; unless otherwise specified, the test materials used in the following examples are all obtained by purchasing from conventional biochemical reagent companies.

[0032] The gene accession number of SLFN12 protein is: NM_018042.

[0033] Example 1 Inhibition of replication of viruses such as EV71, EMCV and SVA by SLFN12 protein

[0034] 1. Construction of overexpressing Flag-SLFN12 plasmid

[0035] The Flag-SLFN12 plasmid was synthesized by Wuhan Kingcare Bioengineering Co., Ltd. Among them, the SLFN12 gene was synthesized according to the human SLFN12 gene sequence, and the gene accession number is: NM_018042. This gene was ligated to the p3×FLAG-CMV-7.1 vector through restriction enzyme sites.

[0036] 2. Sample preparation for overexpressing Flag-SLFN12 plasmid transfected into HEK-293T cells and then infecting with EV71, EMCV and SVA

[0037] HEK-293T cells were cultured in a 12-well cell culture plate. When the cells grew to 60% - 70%, 0 ng, 250 ng and 500 ng of Flag-SLFN12 plasmid were transfected. After 24 h, the cells were gently washed twice with PBS. EV71, EMCV and SVA at 0.1 MOI were respectively inoculated. After 24 h of infection, the cells were collected for detecting the expression of viral mRNA and protein.

[0038] 3. Detection of viral mRNA content

[0039] After overexpressing the Flag-SLFN12 plasmid in HEK-293T cells for 24 hours, the viral mRNA content was detected after infecting with EV71, EMCV and SVA for 24 hours respectively.

[0040] The results are as Figure 1As shown, overexpression of SLFN12 significantly inhibited the replication of EV71, EMCV, and SVA, indicating that SLFN12 can be used as an inhibitor of EV71, EMCV, and SVA virus replication and is useful for preparing drugs against picornavirus infections.

[0041] 4. Detection of viral protein expression levels by Western Blotting

[0042] After overexpressing Flag-SLFN12 in HEK-293T cells for 24 hours and then infecting with EV71 and SVA, Western Blotting was performed to detect the expression levels of viral proteins.

[0043] The detection results of the 3D protein after infecting EV71 and the VP2 protein after infecting SVA in HEK-293T cells overexpressing Flag-SLFN12 for 24 hours are as Figure 2 shown. Overexpression of SLFN12 significantly inhibited the expression of the EV71 non-structural protein 3D and the SVA structural protein VP2, indicating that SLFN12 can significantly inhibit the replication of EV71 and SVA and is useful for preparing drugs against picornavirus infections.

[0044] Example 2 Effect of interfering with the expression of SLFN12 protein on the replication of SVA in HELA cells

[0045] 1. Design of si-RNA

[0046] The SLFN12 siRNA sequences were designed and synthesized by Beijing Tsingke Biotechnology Co., Ltd.

[0047] The siRNA sequences are as follows:

[0048] F: 5’-GAAAGUGUCUCACGAGCUA(dT)(dT)-3’;

[0049] R: 5’-UAGCUCGUGAGACACUUUC(dT)(dT)-3’.

[0050] 2. Effect of interfering with the expression of SLFN12 in HELA cells on the replication of SVA

[0051] HELA cells were cultured in a 12-well cell culture plate. When the cells reached 60% - 70% confluence, they were transfected with siRNA against the SLFN12 protein for 36 hours. After gently washing twice with PBS, 1 MOI of SVA was inoculated. After 24 hours of infection, the cells were collected for detecting the mRNA and protein replication of the virus.

[0052] 3. Detection of SLFN12 mRNA and protein contents

[0053] The SLFN12 quantitative primer sequences were synthesized by Beijing Tsingke Biotechnology Co., Ltd.

[0054] The upstream primer sequence is: tgt ttg cta aag agc ctg att cc;

[0055] The downstream primer sequence is: ttt ggt tca gcc tcc acc at.

[0056] The results were as Figure 3 shown. After interfering with SLFN12 protein in HELA cells for 36 hours, the content of SLFN12 decreased significantly, indicating that the expression of SLFN12 protein was successfully interfered in HELA cells; moreover, compared with the blank control, after interfering with SLFN12 protein in HELA cells and then infecting with SVA for 24 hours, the content of SLFN12 protein also decreased significantly.

[0057] 4. Detection of viral mRNA content and structural protein VP2

[0058] After interfering with SLFN12 protein in HELA cells for 36 hours and then infecting with SVA for 24 hours, the replication of the virus was detected.

[0059] The results were as Figure 4 shown, in which both the SVA mRNA content and the expression of structural protein VP2 were significantly up-regulated, indicating that inhibiting the expression of SLFN12 protein could promote the replication of SVA. Knocking down or knocking out SLFN12 protein in host cells could be used as a production cell line for picornaviridae viruses or virus vaccines to enhance the production of viruses or vaccines.

[0060] Example 3 Inhibition of SVA replication by SLFN12 protein

[0061] 1. Preparation of samples after transfection of HEK-293 cells with overexpressed Flag-SLFN12 plasmid and then infection with SVA

[0062] The method was the same as that described in item 2 of Example 1. The supernatant was collected for measuring the virus titer in the supernatant; the cells were collected for detecting the expression of viral mRNA.

[0063] 2. Detection of viral mRNA content and virus titer

[0064] After transfecting HEK-293T cells with Flag-SLFN12 plasmid for 24 hours and then infecting with 0.1 MOI SVA for 24 hours, the viral mRNA content and virus titer were detected.

[0065] The detection results were as Figure 5As shown, overexpression of SLFN12 significantly inhibited the replication of SVA virus, indicating that SLFN12 can significantly inhibit the replication of SVA.

[0066] 3. Cell fluorescence detection

[0067] Culture HEK-293T cells in a 12-well cell culture plate. When the cells grow to 60% - 70%, transfect with Flag-SLFN12 plasmid for 24 hours. Gently wash twice with PBS and infect with 0.5 MOI SVA-GFP for 24 hours. Detect the cell fluorescence with an inverted fluorescence microscope.

[0068] The detection results are as Figure 6 shown, overexpression of SLFN12 significantly inhibited the replication of SVA virus.

[0069] Example 4 Inhibition of FMDV replication by SLFN12 protein

[0070] 1. Preparation of samples for infecting FMDV after transfecting PK-15 cells with overexpressed Flag-SLFN12 plasmid

[0071] Culture PK-15 cells in a 12-well cell culture plate. When the cells grow to 60% - 70%, transfect with 0 ng, 200 ng, and 400 ng of Flag-SLFN12 plasmid. After 24 h, gently wash the cells twice with PBS. Inoculate with 0.5 MOI of FMDV. After 12 h of infection, collect the cells for detecting the expression of viral mRNA and protein; collect the supernatant for measuring the virus titer in the supernatant.

[0072] 2. Detection of viral mRNA content

[0073] Detect the viral mRNA content 12 hours after infecting FMDV 24 hours after transfecting PK-15 cells with overexpressed Flag-SLFN12 plasmid.

[0074] The results are as Figure 7 shown, overexpression of SLFN12 protein significantly inhibited the replication of FMDV.

[0075] 3. Determination of virus titer

[0076] Detect the virus titer 12 hours after infecting FMDV 24 hours after transfecting PK-15 cells with overexpressed Flag-SLFN12 plasmid.

[0077] The detection results are as Figure 8 shown, overexpression of SLFN12 protein significantly inhibited the replication of FMDV.

[0078] The above results indicate that the SLFN12 protein can significantly inhibit the replication of EV71, EMCV, SVA, and FMDV viruses and can be used to prepare drugs or vaccine adjuvants against Picornaviridae virus infections. Moreover, after interfering with the expression of the SLFN12 protein, it can significantly promote the replication of Picornaviridae viruses. By constructing a SLFN12 protein knockout cell line as a production cell line for Picornaviridae viruses or vaccines, the expression efficiency of Picornaviridae viruses or vaccines can be increased.

Claims

1. Use of SLFN12 protein or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing or treating Picornaviridae virus infection.

2. The use according to claim 1, characterized in that The Picornaviridae virus is selected from EV71, EMCV, SVA, and FMDV.

3. Use of SLFN12 protein or a pharmaceutically acceptable salt thereof in the preparation of Picornaviridae virus vaccine adjuvants.

4. The use according to claim 3, characterized in that The Picornaviridae virus is selected from EV71, EMCV, SVA, and FMDV.

5. Application of SLFN12 protein knockout / knockdown cell line as a production cell line for Picornaviridae viruses or viral vaccines.

6. Application of reagents interfering with / inhibiting SLFN12 protein expression in the preparation of synergists for the production of Picornaviridae viruses or viral vaccines.

7. The use according to claim 5 or 6, characterized in that The Picornaviridae virus is selected from EV71, EMCV, SVA, and FMDV.

8. A siRNA that interferes with the expression of SLFN12 protein, the sequence of the siRNA is: F: 5'-GAAAGUGUCUCACGAGCUA(dT)(dT)-3'; R: 5'-UAGCUCGUGAGACACUUUC(dT)(dT)-3'.

9. Use of the siRNA according to claim 8 in preparing a production cell line of a Picornaviridae virus or a virus vaccine.

10. The use according to claim 9, characterized in that The Picornaviridae virus is selected from EV71, EMCV, SVA, and FMDV.

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