Application of SLC15A3 in preparation of anti-dengue virus medicine

Abstract

The invention relates to an application of SLC15A3 in preparation of an anti-dengue virus drug, and discovers that a novel antiviral protein SLC15A3 has a novel application of efficiently targeting a dengue virus non-structural protein NS4A so as to inhibit the proliferation of a dengue virus in a host cell. The invention develops a new field of clinical application of the humanized antiviral protein in the field of antiviral prevention and treatment, so that a new thought and direction are provided for drug development of targeted dengue virus non-structural protein NS4A, a scientific basis is provided for development of anti-dengue virus drugs, and a new thought is provided for clinical antiviral treatment.

Description

Technical Field

[0001] This application relates to the field of anti-dengue virus technology, and in particular to the application of SLC15A3 in the preparation of anti-dengue virus drugs. Background Technology

[0002] Dengue virus (DENV) belongs to the genus Flaviviridae in the family Flaviviridae, and is a single-sense positive-sense RNA virus. Dengue virus is primarily transmitted by Aedes aegypti mosquitoes (…). Aedes aegypti ) and Aedes albopictus ( Aedes albopictus Transmitted by insects such as larvae, dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS) are caused by the dengue virus. Dengue fever is the most common, an acute infectious disease caused by the dengue virus, characterized by rapid spread and high morbidity, and is classified as a Class B infectious disease. Severe dengue fever (DHF / DSS) is characterized by high fever, severe hemorrhage, shock, and a high mortality rate.

[0003] Currently, dengue fever treatment mainly focuses on symptomatic and supportive therapy, which can only alleviate symptoms and cannot cure the viral infection. Specific anti-dengue virus drugs and vaccines are not yet widely used. Patients mainly rely on their own innate and adaptive immunity to fight off DENV infection. The innate immune system is considered the body's first line of defense against viral infection, and type I interferons (IFN-I) are key cytokines of the innate immune system. Rapid and widespread production of IFN-I by the host is an important event in the host's innate immune system's antiviral response. IFN-I itself does not have a direct antiviral effect, but acts on the type I interferon-α / β receptor (IFNAR) of target cells through autocrine or paracrine mechanisms, thereby activating downstream signaling pathways and ultimately leading to the production of a large number of interferon-stimulated genes (ISGs). Domestic and international studies have shown that dengue virus can evade immune surveillance by regulating the host's interferon response and ISG expression, thereby promoting infection. Furthermore, some ISGs, such as TRIM69, have been shown to inhibit dengue virus replication, demonstrating potential as therapeutic targets. Currently, ISG-based antiviral strategies are rapidly developing, and certain novel ISGs may have the potential to inhibit viral replication and modulate host immune responses. However, their specific antiviral functions and mechanisms of action have not yet been fully elucidated.

[0004] SLC15A3 (Solute Carrier Family 15 Member 3) is located on the long arm of chromosome 11, region 1, band 2, subband 2. The full-length gene is 2506 bp, composed of seven exons and six introns, with two transcriptomic splice variants. The expressed protein product contains 581 amino acid residues. SLC15A3 belongs to the mammalian proton-coupled oligopeptide transporter family, also known as OCTP, PHT2, PTR3, or hPHT2. Current research has found that SLC15A3 plays an important role in immune regulation; activation of upstream NF-κB and IRF3 in the interferon pathway upregulates SLC15A3 expression, indicating that its function as an ISG is regulated by upstream interferon. Currently, research on the antiviral function of SLC15A3 is limited, with only its ability to inhibit herpes simplex virus type 1 (HSV-1) found. Longzhen et al. found that SLC15A3 can bind to MAVS and STING and promote interferon expression. This suggests that SLC15A3 may have a positive feedback regulation on upstream IFN production. SLC15A3 can also promote the degradation of SQSTM1 / p62 via ubiquitination and autophagy, thereby promoting oxidative stress in macrophages. This suggests that SLC15A3 may also participate in immune regulation by influencing the autophagy pathway. Furthermore, SLC15A3 is widely distributed on the membranes of vascular endothelial cells and astrocytes, and affects transcellular transport and astrocyte activation under stress. Although SLC15A3 can participate in the innate immune response through multiple pathways, its biological function of inhibiting dengue virus replication and its antiviral mechanism are currently unknown. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of SLC15A3 in the preparation of anti-dengue virus drugs.

[0006] The first objective of this invention is to provide the use of protein SLC15A3 in the preparation of dengue virus non-structural protein inhibitors.

[0007] A second objective of this invention is to provide the use of protein SLC15A3 in the preparation of drugs against dengue virus.

[0008] A third objective of this invention is to provide the use of protein SLC15A3 in the preparation of medicaments for treating diseases caused by dengue virus.

[0009] The fourth object of the present invention is to provide a drug for treating dengue virus or dengue-induced diseases.

[0010] To achieve the above objectives, the present invention is implemented through the following technical solution: This invention discovers that SLC15A3 (Gene ID: 51296, GenBank accession number: NM_016582.3) has the ability to inhibit dengue virus at the cellular level, indicating that it can serve as a novel drug for inhibiting dengue virus. Specifically, this invention uses immunoblotting to demonstrate that dengue virus infection can significantly induce an increase in the expression level of SLC15A3 protein in host cells; uses a stable expression system and immunoblotting to demonstrate that SLC15A3 can inhibit dengue virus replication in cells; and uses immunoprecipitation experiments to confirm the interaction between SLC15A3 and the dengue virus non-structural protein NS4A.

[0011] This invention claims the use of the protective protein SLC15A3 in the preparation of dengue virus non-structural protein inhibitors.

[0012] Preferably, the dengue virus non-structural protein is the non-structural protein NS4A.

[0013] The present invention also claims the use of the protective protein SLC15A3 in the preparation of drugs against dengue virus.

[0014] And the use of protein SLC15A3 in the preparation of drugs for treating dengue virus-induced diseases.

[0015] Preferably, the disease is dengue fever, dengue hemorrhagic fever, or dengue shock syndrome.

[0016] Preferably, protein SLC15A3 forms a complex with dengue virus non-structural proteins.

[0017] Preferably, the dengue virus non-structural protein is the non-structural protein NS4A.

[0018] Preferably, the protein SLC15A3 inhibits dengue virus replication in host cells.

[0019] Preferably, the drug is in the form of tablets, oral liquids, oral pills, oral granules, oral powders, injections, eye drops, nasal drops, aerosols, or inhalers.

[0020] This invention also claims protection for a drug for treating dengue virus or dengue-induced disease, containing the protein SLC15A3.

[0021] Compared with the prior art, the present invention has the following beneficial effects: This invention discovers a novel application of the antiviral protein SLC15A3, which efficiently targets the dengue virus non-structural protein NS4A, thereby inhibiting dengue virus proliferation in host cells. This opens up a new field of clinical application for humanized antiviral proteins in antiviral prevention and treatment, providing new ideas and directions for drug development targeting the dengue virus non-structural protein NS4A, providing a scientific basis for the development of anti-dengue drugs, and offering new ideas for clinical antiviral therapy. Attached Figure Description

[0022] Figure 1 The figure shows the results of using real-time quantitative RT-PCR to demonstrate that both IFN-α and IFN-β stimulation can significantly induce an increase in the mRNA level of SLC15A3 in A549 cells.

[0023] Figure 2 The figure shows the results of using Western blotting to demonstrate that dengue virus infection can significantly induce an increase in the expression level of SLC15A3 protein in A549 cells.

[0024] Figure 3 The figure shows the results of using an exogenous protein expression system and Western blotting to demonstrate that SLC15A3 can inhibit dengue virus replication in A549 cells.

[0025] Figure 4 The figure shows the results of an immunoprecipitation experiment demonstrating the interaction between SLC15A3 and the dengue virus non-structural protein NS4A. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0027] Example 1: IFN-α and IFN-β stimulation significantly induced an increase in SLC15A3 mRNA levels. I. Experimental Methods 1. A suitable amount of A549 cells were seeded into cell culture dishes. After 24 hours, the cells were stimulated with 500 U / ml of type I interferon IFN-α and IFN-β, respectively. Cells were collected at four time points after stimulation: 6 h, 12 h, 18 h, and 24 h, for subsequent real-time quantitative PCR analysis.

[0028] 2. The specific operation of real-time quantitative PCR is as follows: (1) Total RNA was extracted using the TRIzol method. First, the cell culture medium was discarded and the cells were gently washed twice with pre-cooled PBS. Then, an appropriate amount of TRIzol lysis buffer was added to the culture dish to fully lyse the cells, and the lysis buffer was transferred to an RNase-free centrifuge tube and allowed to stand at room temperature for 5 minutes. Next, chloroform was added in proportion and shaken vigorously to form an emulsion. After standing at room temperature, the emulsion was centrifuged at 12000×g for 15 minutes at 4°C. The colorless aqueous phase was carefully aspirated into a new centrifuge tube, and an equal volume of isopropanol was added to precipitate RNA at room temperature for 10 minutes. After centrifugation again, a white RNA precipitate was obtained. Finally, the precipitate was washed with 75% ethanol, dried briefly, and then RNase-free water was added to dissolve the RNA. The RNA concentration and purity were detected by a micro spectrophotometer to ensure that the A260 / A280 ratio was between 1.8 and 2.1.

[0029] (2) Take 1 μg of total RNA, add reverse transcription reagent, mix gently and place in a PCR instrument to run the reverse transcription program. The resulting cDNA product can be used immediately for subsequent experiments or frozen at -20℃.

[0030] (3) Prepare the real-time quantitative PCR reaction system on ice in an optical grade 96-well plate (see Table 1). The primers for gene detection are shown in Table 2. After adding the samples, gently pipette to mix, briefly centrifuge to collect the droplets on the tube wall, and immediately seal the plate opening tightly with an optical transparent sealing film.

[0031] Table 1. Real-time quantitative PCR reaction system

[0032] Table 2. Primers related to gene detection

[0033] (4) Place the sealed reaction plate in a real-time quantitative PCR instrument, set the amplification program, and perform melting curve analysis at the end of the program to verify the specificity of the amplification product and exclude non-specific amplification.

[0034] II. Experimental Results The results are as follows Figure 1 As shown, real-time quantitative PCR experiments revealed that both IFN-α and IFN-β stimulation significantly induced the upregulation of SLC15A3 mRNA levels in a time-dependent manner.

[0035] Example 2: Dengue virus infection induces increased expression levels of SLC15A3 protein in host cells. I. Experimental Methods 1. Sow an appropriate amount of dengue virus-susceptible cells A549 (human lung adenocarcinoma cell line) and Huh7 (human liver cancer cell line) into a 10cm cell culture dish. After 24 hours, infect the cells with dengue virus (MOI=2). After 24 hours, extract the total protein of the cells and detect the corresponding proteins by immunoblotting.

[0036] 2. Detection of SLC15A3 protein expression by Western blotting: Perform reducing SDS-PAGE on the protein sample. Stop electrophoresis after bromophenol blue has eluted the gel. Prepare two 3M filter papers and one PVDF membrane, immerse them in methanol-deionized water for 5 minutes, then soak them in 1× transfer buffer along with the special filter paper and fiber pad. Peel off the gel, remove the stacking gel portion, and cut the filter paper and PVDF membrane to the gel size. Transfer the membrane using the sandwich method, with the following arrangement: negative electrode – fiber pad – 1 special filter paper – gel – PVDF membrane – 1 special filter paper – fiber pad positive electrode plate. Place it in the transfer tank. Transfer at 300 mA for 2 hours on ice. Remove the PVDF membrane and block it with blocking buffer for 1 hour. Incubate overnight at 4°C with SLC15A3 primary antibody. Recover the primary antibody, wash the membrane with TBST for 10 minutes each time, repeating 3 times. Incubate with secondary antibody at room temperature for 60 minutes. Recover the secondary antibody, wash the membrane with TBST for 10 minutes each time, repeating 3 times. Develop with ECL chemiluminescence in a darkroom.

[0037] II. Experimental Results The results are as follows Figure 2 As shown, immunoblotting experiments indicate that dengue virus infection can significantly induce an increase in the expression level of SLC15A3 protein in host cells.

[0038] Example 3: Inhibitory effect of SLC15A3 protein on dengue virus infection I. Experimental Methods 1. Construction and identification of recombinant plasmids pSin-SLC15A3-HA and pSin-DENV2-NS4A-Flag (1) Reverse transcription reaction cDNA was synthesized using total RNA from 293T cells as a template via reverse transcription.

[0039] (2) Primer design and PCR amplification The SLC15A3 gene sequence was downloaded from the GenBank database (GenBank accession number: NM_016582.3). Primers were designed to amplify the full-length ORF fragment of the SLC15A3 gene, and then HA tags were introduced into the product of SLC15A3 amplification using primers. SLC15A3-HA ).

[0040] Primers were designed to amplify the full-length ORF fragment of the SLC15A3 gene. The NS4A gene sequence of the dengue virus type 2 NGC standard strain (DENV2-NGC, GenBank accession number: AF038403) (DENV2-NGC virus strain is preserved by the inventor's research group) was then amplified. A flag tag was introduced into the product of the amplified DENV2-NS4A gene fragment. DENV2-NS4A-Flag ).

[0041] Primers were synthesized by Shanghai Yingjun Biotechnology Co., Ltd., and their sequences are shown in Table 3.

[0042] Table 3. Primers related to gene cloning

[0043] (3) Plasmid construction and ligation PCR amplification SLC15A3-HA and DENV2-NS4A-Flag The plasmid was subjected to double enzyme digestion with the pSin-Vector vector, followed by ligation with T4 DNA ligase. The ligation product was extracted from Escherichia coli competent cells DH5α using a rapid plasmid miniprep kit (DP105, Tiangen Biotech (Beijing) Co., Ltd.).

[0044] (4) Transformation and screening The ligation product was added to 200 μl of DH5α competent bacteria for transformation. A single colony was picked and inoculated into LB medium containing ampicillin (50 μg / ml) and incubated overnight at 37 °C.

[0045] (5) Plasmid extraction and identification Recombinant plasmid DNA from pSin-SLC15A3-HA and pSin-DENV2-NS4A-Flag was extracted using a rapid plasmid miniprep kit (DP105, Tiangen Biotech (Beijing) Co., Ltd.). The correctness of plasmid construction was verified by enzyme digestion and sequencing. Sequencing results were compared and analyzed using NCBI's BLAST program, confirming that the constructed plasmids were 100% homologous to the SLC15A3 and DENV2-NS4A sequences registered in GenBank.

[0046] 2. The method for constructing a stable cell line with high expression of SLC15A3 protein is as follows: (1) The pSin-Vector and pSin-SLC15A3-HA plasmids were transfected into 293T cells using liposome transfection. (2) Collect viral supernatant carrying the SLC15A3 gene and use it to infect A549 cells; (3) Through puromycin screening and continuous passage culture, A549 cell line (A549-SLC15A3 cell) that stably expresses SLC15A3 protein was finally obtained. (4) In the fifth generation of cells, total cell protein was extracted and the expression level of SLC15A3 protein was detected by immunoblotting.

[0047] 3. A suitable amount of A549-SLC15A3 and A549-Vector cells were seeded into cell culture dishes P100. After 24 hours, they were infected with dengue virus (MOI=0.5). After 48 hours, total cell protein was extracted and the corresponding proteins were detected by immunoblotting.

[0048] 4. Immunoblotting detection of DENV-2 NS4A protein levels: Perform reducing SDS-PAGE on the protein sample. Stop electrophoresis after bromophenol blue has eluted the gel. Prepare two 3M filter papers and one PVDF membrane, immerse them in methanol-deionized water for 5 minutes, then soak them in 1× transfer buffer along with the special filter paper and fiber pad. Peel off the gel, remove the stacking gel portion, and cut the filter paper and PVDF membrane to the gel size. Transfer the membrane using the sandwich method, with the following arrangement: negative electrode – fiber pad – 1 special filter paper – gel – PVDF membrane – 1 special filter paper – fiber pad positive electrode plate. Place it in the transfer tank. Transfer at 300 mA for 2 hours on ice. Remove the PVDF membrane and block it with blocking buffer for 1 hour. Incubate with NS4A primary antibody overnight at 4°C. Recover the primary antibody, wash the membrane with TBST for 10 minutes each time, repeating 3 times. Incubate with secondary antibody at room temperature for 60 minutes. Recover the secondary antibody, wash the membrane with TBST for 10 minutes each time, repeating 3 times. Develop with ECL in a darkroom.

[0049] II. Experimental Results See results Figure 3 As shown, compared with A549 cells transfected with pSin-Vector (A549-Vector), the NS4A protein level of DENV2 in A549-SLC15A3 cells was significantly reduced, indicating that exogenous high expression of SLC15A3 protein can effectively inhibit dengue virus replication in host cells.

[0050] Example 4: The host SLC15A3 protein and the dengue virus non-structural protein NS4A bind to each other. I. Experimental Methods 1. Cell transfection (1) Using 293T cells as a cell model, the cells were seeded 24 h before transfection; (2) Add 200 ng each of the plasmid pSin-SLC15A3-HA (high expression of SLC15A3) and pSin-DENV2-NS4A-Flag (high expression of NS4A) to 25 μl Opti-MEM, and add P3000. TM 1 μl, gently mix; (3) Add 2 μl of Lipofectamine to 25 μl of Opti-MEM. TM Mix 3000 reagents gently and let stand at room temperature for 5 minutes; (4) Mix the liquids in (2) and (3) above, gently tap the tube wall to mix, and let stand at room temperature for 5 min; (5) Add (4) to the culture medium of 293T cells and incubate in a 37°C incubator; (6) Change the medium after 4-6 h and continue culturing to obtain 293T cells that highly express SLC15A3 and 293T cells that highly express NS4A. 2. Immunoprecipitation assay to detect the interaction between host SLC15A3 protein and dengue virus non-structural protein NS4A. The specific method is as follows: (1) The 293T cells with high expression of SLC15A3 and 293T cells with high expression of NS4A were prepared, and the original culture medium was discarded. They were washed twice with pre-cooled 1×PBS. (2) Add 6 ml of pre-cooled 1×PBS, scrape the cells from the culture dish, and transfer the cell solution to a 15 ml centrifuge tube. Centrifuge at 2000 rpm for 4 ℃ for 5 min. (3) Discard the supernatant, add 500 μl of 1×IP lysis buffer (containing protease inhibitor) to the cell pellet, and transfer the cell suspension to a 1.5 ml centrifuge tube. Place the tube on ice and shake it every 10 min for 30 s each time, for a total of 30 min to ensure that the cells are fully lysed. (4) After centrifuging at 12000 rpm for 30 min, take the supernatant and repeat this step once; (5) Take 30 μl of lysis buffer as input, add HA magnetic beads and negative control IgG magnetic beads to the remaining lysis buffer for pull-down experiment, and incubate overnight at 4°C with slow shaking; (6) Wash the magnetic beads 5-6 times with 1×IP wash buffer. Be careful to be gentle to avoid damaging the protein binding. (7) Add 30 μl of 1×sample buffer to the magnetic bead-protein mixture, add 10 μl of 4×sample buffer to the input, and heat in a 100℃ metal bath for 10 min.

[0051] 3. Immunoblotting detection of proteins with HA or Flag tags Perform reducing SDS-PAGE on the protein sample. Stop electrophoresis after bromophenol blue has eluted the gel. Prepare two 3M filter papers and one PVDF membrane, immerse them in methanol-deionized water for 5 minutes, then soak them in 1× transfer buffer along with the special filter paper and fiber pad. Peel off the gel, remove the stacking gel portion, and cut the filter paper and PVDF membrane to the gel size. Transfer the membrane using the sandwich method, with the following arrangement: negative electrode – fiber pad – 1 special filter paper – gel – PVDF membrane – 1 special filter paper – fiber pad positive electrode plate. Place it in the transfer tank. Transfer at 300 mA for 2 hours on ice. Remove the PVDF membrane and block it with blocking buffer for 1 hour. Incubate overnight at 4°C with primary antibody (HA antibody or Flag antibody). Recover the primary antibody, wash the membrane with TBST for 10 minutes each time, repeating 3 times. Incubate with secondary antibody at room temperature for 60 minutes. Recover the secondary antibody, wash the membrane with TBST for 10 minutes each time, repeating 3 times. Develop with ECL in a darkroom.

[0052] II. Experimental Results See results Figure 4 As shown, the exogenous SLC15A3 protein forms a complex with the NS4A protein of DENV2, further demonstrating that the two bind to each other, thus proving that SLC15A3 targets and inhibits the NS4A protein of dengue virus.

Claims

1. Application of protein SLC15A3 in the preparation of dengue virus non-structural protein inhibitors.

2. The application according to claim 1, characterized in that, The dengue virus non-structural protein is non-structural protein NS4A.

3. Application of protein SLC15A3 in the preparation of drugs against dengue virus.

4. The use of protein SLC15A3 in the preparation of drugs for treating dengue virus-induced diseases.

5. The application according to claim 4, characterized in that, The disease in question is dengue fever, dengue hemorrhagic fever, or dengue shock syndrome.

6. The application according to claim 3 or 4, characterized in that, The protein SLC15A3 forms a complex with dengue virus non-structural proteins.

7. The application according to claim 6, characterized in that, The dengue virus non-structural protein is non-structural protein NS4A.

8. The application according to claim 3 or 4, characterized in that, The protein SLC15A3 inhibits dengue virus replication in host cells.

9. The application according to claim 3 or 4, characterized in that, The drug is in the form of tablets, oral liquids, oral pills, oral granules, oral powders, injections, eye drops, nasal drops, aerosols, or inhalers.

10. A drug for treating dengue virus or dengue-induced diseases, characterized in that, It contains the protein SLC15A3.

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