Use of slfn12 protein in the preparation of a medicament against a picornaviridae virus

Abstract

The application belongs to the field of biotechnology, and particularly relates to application of SLFN12 protein in preparation of medicine for resisting viruses of Picornaviridae. The application surprisingly finds that, by adding SLFN12 protein in culture medium for culturing EV71, EMCV, SVA and FMDV, the replication level of EV71, EMCV, SVA and FMDV is reduced, which indicates that the SLFN12 protein has the function of inhibiting the replication of viruses of Picornaviridae, and can be used for preparing medicine or adjuvant for resisting virus infection, and for inhibiting the replication of viruses of Picornaviridae. Secondly, the cell line after interfering with the expression of SLFN12 protein in host cells can significantly promote the replication of viruses of Picornaviridae, and can be used as a production cell line for viruses of Picornaviridae or viral vaccine, and for expression production of viruses of Picornaviridae or viral vaccine.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biotechnology, and particularly relates to application of SLFN12 protein in preparation of medicine for resisting viruses of Picornaviridae. BACKGROUND

[0002] Picornaviridae is a family of the smallest group of RNA viruses. The viruses of this family are a class of single-stranded positive-strand RNA viruses widely existing in humans and animals, which can cause a variety of serious infectious diseases, and pose a serious threat to human health and animal husbandry. Representative viruses include enterovirus 71 (EV71) in Enterovirus, encephalomyocarditis virus (EMCV) in Cardiovirus, Seneca Valley virus (SVA) in Senecavirus, and foot-and-mouth disease virus (FMDV) in Aphthovirus, etc. EV71 is a small RNA virus that mainly infects children and can cause a variety of diseases; EMCV is a small RNA virus that is pathogenic to a variety of animals and humans; both have high pathogenicity, especially in terms of neurological and myocardial damage. Foot-and-mouth disease is a highly contagious, acute febrile animal infectious disease caused by foot-and-mouth disease virus, mainly infecting odd-toed animals (such as pigs, cattle, sheep, etc.), causing serious threats to global animal husbandry, and remaining difficult to prevent; SVA is a pathogen that has attracted attention in pig populations in recent years and can cause vesicular disease in pigs; FMDV and SVA are both pathogens that pose a significant threat to animal husbandry, with similar clinical symptoms, and pose a huge threat to the pig industry, making the prevention and control of the disease extremely severe.

[0003] SLFN12 protein is an important member of the Schlafen family of proteins, and this family member is associated with 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-damaging drugs. SLFN12 is expressed in monocytes, monocyte-derived dendritic cells, and T cells. In recent years, SLFN12 protein has attracted widespread attention in the fields of tumor and immunotherapy. The potential role of SLFN12 in the tumor microenvironment. SLFN12 can promote the proliferation, migration of glioma cells and the recruitment of macrophages, thereby affecting tumor progression and immunotherapy effect. 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

[0004] In view of the above technical problems, the application provides application of SLFN12 protein in preparation of medicine for resisting viruses of Picornaviridae. Specifically, the following content is included:

[0005] In a first aspect, the present application provides a use of a SLFN12 protein or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing or treating a Picornaviridae virus infection.

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

[0007] Preferably, the SLFN12 protein or the pharmaceutically acceptable salt thereof is added into a pharmaceutically acceptable carrier and / or adjuvant to form any pharmaceutically acceptable dosage form.

[0008] Preferably, the dosage form includes powder injection, capsule, tablet, suspension.

[0009] In a second aspect, the present application provides a use of a SLFN12 protein or a pharmaceutically acceptable salt thereof in the preparation of a Picornaviridae virus vaccine adjuvant.

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

[0011] Preferably, the SLFN12 protein or the pharmaceutically acceptable salt thereof is added into a pharmaceutically acceptable carrier and / or adjuvant to form any pharmaceutically acceptable dosage form.

[0012] Preferably, the dosage form includes powder injection, capsule, tablet, suspension.

[0013] In a third aspect, the present application provides a use of a SLFN12 protein knock-out / knock-down cell line as a production cell line of a Picornaviridae virus or a virus vaccine.

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

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

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

[0017] In a fifth aspect, the present application provides a siRNA interfering with the expression of a SLFN12 protein, and the sequence of the siRNA is as follows:

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

[0019] R: 5'-UAGCUCGUGAGACACUUUC(dT)(dT)-3'.

[0020] In a sixth aspect, the present application provides the use of the siRNA according to the fifth aspect in the preparation of a production cell line of a virus of the Picornaviridae family or a viral vaccine.

[0021] Preferably, the virus of the Picornaviridae family is selected from EV71, EMCV, SVA, FMDV.

[0022] The beneficial effects of the present application are: the present application surprisingly found that adding SLFN12 protein in the culture medium of EV71, EMCV, SVA, FMDV can reduce the replication level of EV71, EMCV, SVA, FMDV, indicating that SLFN12 protein has the effect of inhibiting the replication of viruses of the Picornaviridae family, and can be used for preparing an antiviral infection drug or adjuvant, and for inhibiting the replication of viruses of the Picornaviridae family; secondly, the cell line after interfering with the expression of SLFN12 protein in the host cell can significantly promote the replication of viruses of the Picornaviridae family, and can be used as a production cell line of a virus of the Picornaviridae family or a viral vaccine, and for the expression production of viruses of the Picornaviridae family or a viral vaccine. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Detection results of viral mRNA content after 24 hours of overexpression of Flag-SLFN12 in HEK-293 cells and infection of EV71, EMCV and SVA;

[0024] Figure 2 Detection results of 3D protein after 24 hours of overexpression of Flag-SLFN12 in HEK-293 cells and infection of EV71, and detection results of VP2 protein after infection of SVA;

[0025] Figure 3 Detection results of SLFN12 after 36 hours of interference of SLFN12 in HELA cells and infection of SVA;

[0026] Figure 4 Detection results of viral mRNA content and VP2 protein after 36 hours of interference of SLFN12 in HELA cells and infection of SVA;

[0027] Figure 5 Detection results of viral mRNA content and viral titer after 12 hours of overexpression of Flag-SLFN12 in HEK-293 cells and infection of SVA;

[0028] Figure 6 Detection results of viral fluorescence after 12 hours of overexpression of Flag-SLFN12 in HEK-293 cells and infection of SVA;

[0029] Figure 7 Results of detection of viral mRNA content after FMDV infection in PK-15 cells overexpressing SLFN12 for 24 hours;

[0030] Figure 8 Results of detection of viral titer after FMDV infection in PK-15 cells overexpressing SLFN12 for 24 hours. DETAILED DESCRIPTION

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

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

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

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

[0035] The Flag-SLFN12 plasmid was synthesized by Wuhan Jin Kai Rui Biological Engineering Co., Ltd., wherein the SLFN12 gene was synthesized according to the sequence of the human SLFN12 gene, and the gene accession number is: NM_018042. The gene was connected to the p3xFLAG-CMV-7.1 vector through enzyme digestion sites.

[0036] 2. Preparation of samples of EV71, EMCV and SVA infection after transfection of HEK-293T cells with overexpression Flag-SLFN12 plasmid

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

[0038] 3. Detection of viral mRNA content

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

[0040] The results are as follows Figure 1As shown in FIG. 2, 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 viral replication for the preparation of a drug against Picornaviridae virus infection.

[0041] 4. Western Blotting technique for determining the expression amount of viral protein

[0042] Western Blotting technique for determining the expression amount of viral protein was performed after EV71 and SVA infection after 24 hours of overexpression of Flag-SLFN12 in HEK-293T cells.

[0043] The results of 3D protein detection after EV71 infection and the results of VP2 protein detection after SVA infection after 24 hours of overexpression of Flag-SLFN12 in HEK-293T cells are shown in FIG. 3. Figure 2 As shown in FIG. 3, overexpression of SLFN12 significantly inhibited the expression of EV71 non-structural protein 3D and the expression of SVA structural protein VP2, indicating that SLFN12 can significantly inhibit the replication of EV71 and SVA and can be used for the preparation of a drug against Picornaviridae virus infection.

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

[0045] 1. Design of si-RNA

[0046] The SLFN12 siRNA sequence was designed and synthesized by Beijing Genki Biotechnology Co., Ltd.

[0047] The siRNA sequence is:

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

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

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

[0051] HELA cells were cultured in a 12-well cell culture plate, and when the cells grew to 60% to 70%, siRNA against SLFN12 protein was transfected for 36 hours. The cells were washed with PBS for 2 times, inoculated with 1 MOI of SVA, and after 24 hours of infection, the cells were collected for detection of viral mRNA and protein replication.

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

[0053] SLFN12 quantitative primer sequence was synthesized by Beijing Qianke 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 are shown in Figure 3 After 36 hours of interference in HELA cells, the content of SLFN12 protein was significantly reduced, indicating that the expression of SLFN12 protein in HELA cells was successfully interfered; and compared with the blank control, after 24 hours of infection of SVA after interfering SLFN12 protein in HELA cells, the content of SLFN12 protein was also significantly reduced.

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

[0058] After 36 hours of interference in HELA cells, the replication of virus was detected after 24 hours of infection of SVA.

[0059] The results are shown in Figure 4 The expression of SVA mRNA content and structural protein VP2 was significantly up-regulated, indicating that the inhibition of SLFN12 protein expression could promote the replication of SVA, and the knockdown or knock-out of SLFN12 protein in host cells could be used as a production cell line of small RNA virus family virus or virus vaccine, for the production of virus or vaccine.

[0060] Example 3: SLFN12 protein inhibits SVA replication

[0061] 1. Sample preparation of SVA infection after transfection of HEK-293 cells by overexpression of Flag-SLFN12 plasmid

[0062] The method is the same as described in 2 of Example 1. The supernatant was collected for determination of the virus titer in the supernatant; the cells were collected for detection of the expression of viral mRNA.

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

[0064] After 24 hours of transfection of Flag-SLFN12 plasmid in HEK-293T cells, the viral mRNA content and virus titer were detected after 24 hours of infection of 0.1 MOI SVA.

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

[0066] 3. Cell fluorescence detection

[0067] HEK-293T cells were cultured in a 12-well cell culture plate, and when the cells grew to 60%-70%, Flag-SLFN12 plasmid was transfected for 24 hours. The cells were washed twice with PBS, and infected with 0.5 MOI SVA-GFP for 24 hours. The cell fluorescence was detected by inverted fluorescence microscopy.

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

[0069] Example 4: SLFN12 protein inhibits FMDV replication

[0070] 1. Preparation of samples of PK-15 cells infected with FMDV after transfection with overexpression Flag-SLFN12 plasmid

[0071] PK-15 cells were cultured in a 12-well cell culture plate, and when the cells grew to 60%-70%, Flag-SLFN12 plasmid was transfected at 0 ng, 200 ng and 400 ng. After 24 hours, the cells were washed twice with PBS. 0.5 MOI of FMDV was inoculated. After 12 hours of infection, the cells were collected for detection of viral mRNA and protein expression; the supernatant was collected for determination of viral titer in the supernatant.

[0072] 2. Detection of viral mRNA content

[0073] After transfection of overexpression Flag-SLFN12 plasmid in PK-15 cells for 24 hours, FMDV was infected for 12 hours to detect the viral mRNA content.

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

[0075] 3. Determination of viral titer

[0076] After transfection of overexpression Flag-SLFN12 plasmid in PK-15 cells for 24 hours, FMDV was infected for 12 hours to detect the viral titer.

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

[0078] The results show that the SLFN12 protein can significantly inhibit the replication of EV71, EMCV, SVA and FMDV viruses, and can be used for preparing a medicine or a vaccine adjuvant against the infection of viruses in the Picornaviridae family. Moreover, the expression of the SLFN12 protein can be significantly promoted after the interference of the SLFN12 protein, and the expression of viruses or vaccines in the Picornaviridae family can be enhanced by constructing a SLFN12 protein knockout cell line as a production cell line of viruses or vaccines in the Picornaviridae family.

Claims

1. Use of SLFN12 protein in the preparation of a drug for treating a Picornaviridae virus infection; the Picornaviridae virus is selected from EV71, EMCV, SVA and FMDV, the gene accession number of the SLFN12 protein is NM_018042.

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