Application of neurodylin-2 in preparation of medicine for preventing and treating chikungunya virus infection

Abstract

The invention relates to the technical field of biomedicine, in particular to a novel target spot for resisting chikungunya virus infection and application. According to the invention, human neuroblastoma cells (SH-SY5Y) and human brain astroblastoma cells (U87) are taken as target cells, and the expression of the target cell part capable of exerting receptor action membrane protein is down-regulated by adopting an RNA interference technology, so that a host factor capable of effectively inhibiting CHIKV infection of human nerve cells is found, and the purpose of blocking CHIKV infection from the source is achieved. The invention discovers that the nerve cilia protein 2 (NRP2) plays an important role in CHIKV infection SH-SY5Y and U87 cells, and the CHIKV infection can be obviously inhibited by down-regulating the expression of the NRP2. The invention provides an application of NRP2 in preparation of a medicine for preventing or treating chikungunya virus infection, and provides a new target spot and a treatment scheme for clinically preventing and treating fever, arthralgia, arthrocele, muscular pain, headache, nausea, fatigue, rash and other diseases caused by CHIKV infection.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a novel target for combating Chikungunya virus infection and its application. Background Technology

[0002] Chikungunya fever (CHIKF) is a vector-borne infectious disease caused by chikungunya virus (CHIKV). Typical clinical manifestations include fever, rash, joint pain, and myalgia. CHIKV belongs to the family Hypoviridae, genus Alphavirus. The virus particles are spherical, 65 nm in diameter, and enveloped.

[0003] The acute phase of CHIKV infection is characterized by persistent high fever, accompanied by significant viremia and a strong innate immune response, a process that can last up to a week. Following the onset of fever, most patients progress to severe bilateral symmetrical polyarthritis, primarily affecting distal joints such as the wrist and ankle, as well as small joints of the hand. Although viremia usually clears within 8 days of infection, and most acute symptoms subside within one month, epidemiological studies show that some individuals develop persistent arthritis and joint pain, which can last from months to years. Patients with underlying chronic diseases are more likely to develop a chronic disease course, significantly impacting their quality of life and imposing a severe economic burden.

[0004] However, there are currently no specific and highly effective antiviral drugs or effective vaccines approved for clinical use for the treatment of CHIKV. The vast majority of patients still receive symptomatic treatment, partly because of the limited understanding of the pathogenesis of CHIKV, and multiple factors may play important roles in its infection process.

[0005] Matrix remodeling associated 8 (MXRA8) is generally considered to be the receptor for various arthritic alphaviruses, including CHIKV. However, other host factors also play crucial roles in CHIKV infection. For example, overexpression of T-cell immunoglobulin and mucin domain protein-1 (TIM-1) in HEK293T cells significantly enhances the binding and endocytosis of CHIKV viral particles. Prohibitin (PHB) has been confirmed to affect CHIKV infection of host cells through CO-IP, siRNA knockdown, and antibody blocking experiments. Recent genome-wide CRISPR-Cas9 screening has identified FHL1 as a key host factor required for CHIKV infection, and knockout of FHL1A significantly promotes both CHIKV and ONNV infection. Furthermore, FHL1 plays a crucial role primarily during the CHIKV replication phase. Therefore, screening out host factors that can influence viral infection can provide a foundation for research on the pathogenesis of CHIKV and the development of specific antiviral drugs.

[0006] Neuropilins (NRPs) are non-tyrosine kinase transmembrane receptor proteins whose gene sequences are highly conserved across all vertebrates. As receptors for serotonins, which act as axonal guidance factors, neuropilins not only regulate neural development but also interact with VEGF to mediate the development of blood and lymphatic vessels. NRPs include two isoforms: neuropilin 1 (NRP1) and neuropilin 2 (NRP2), which can form homodimers and heterodimers and are highly conserved in vertebrates. Current research indicates that both NRP2 and NRP1 can act as receptors for SARS-CoV-2 infection. Furthermore, NRP2 is also a receptor for human cytomegalovirus (HCMV), rabies virus (RABV), and Lujo virus (LUJV).

[0007] However, there are currently no reports on the role of NRP2 molecules in CHIKV infection of host cells. In-depth research on this molecule can not only improve our understanding of the mechanisms of CHIKV infection and pathogenesis, but also provide new ideas and targets for the prevention and treatment of CHIKV infection. Summary of the Invention

[0008] The purpose of this invention is to provide a novel target for resistance to Chikungunya virus infection, namely neuropilin-2 (NRP2).

[0009] Another object of the present invention is to provide new uses for the neurocilia protein 2 (NRP2) molecule, particularly in the treatment of chikungunya virus infection.

[0010] A third objective of this invention is to provide siRNA that interferes with the expression of neuropilin 2 (NRP2) molecules and its applications.

[0011] To achieve the above objectives, the main technical solution of the present invention is as follows:

[0012] This invention uses human neuroblastoma cells (SH-SY5Y) and human astroblastoma cells (U87) as target cells. It employs RNA interference technology to downregulate the expression of receptor-acting membrane proteins in these target cells, aiming to identify host factors that can effectively inhibit CHIKV infection of these cells and thus block CHIKV infection at its source. This invention selects a group of molecules that act as receptors on neurons and glial cells for screening; these molecules may also be easily hijacked and utilized by viruses during infection. These molecules include: AXL receptor tyrosine kinase (AXL), leukemia inhibitory factor receptor (LIFR), pyruvin B1 (PLXNB1), solute carrier family 3 member 2 (SLC3A2), transforming growth factor β receptor 3 (TGFBR3), adhesion G protein-coupled receptor B1 (ADGRB1), ciliary neurotrophic factor receptor (CNTFR), island guide receptor 3 (ROBO3), adhesion G protein-coupled receptor L3 (ADGRL3), adrenaline receptor α1B (ADRA1B), CD44, dipeptidyl peptidase 4 (DPP4), galactopeptide receptor 2 (GALR2), G protein subunit β1 (GNB1), glycoprotein VI (GP6), glutamate ionotropic receptor ruminoid subunit 3 (GRIK3), glutamate ionotropic receptor NMDA type subunit 1 (GRIN1), glutamate ionotropic receptor NMDA type subunit 2A (GRIN2A), and glutamate ionotropic receptor NMDA. The gene sequences identified included subunit 2C (GRIN2C), serotonin receptor 1E (HTR1E), nuclear receptor subfamily 2C member 1 (NR2C1), CD81, kinase receptor 2 (PROKR2), U-type protein tyrosine phosphatase receptor (PTPRU), TYRO3 protein tyrosine kinase, dystroglycan 1 (DAG1), epidermal growth factor receptor (EGFR), tyrosine protein kinase FYN, heat shock protein family B (small) member 1 (HSPB1), integrin subunit β4 (ITGB4), and G protein-coupled receptor 6 containing leucine-rich repetitive sequences (LGR6). Full-length and mRNA sequences were obtained by searching NCBI GeneBank. These genes were then subjected to biological analysis using existing online resources and commonly used software. The coding regions were selected as target sequences for siRNA design. siRNAs were then designed, and the effects on CHIKV infection were observed by downregulating these molecules.

[0013] This invention has shown through experiments that neurocilia protein 2 (NRP2) plays an important role in CHIKV infection of SH-SY5Y and U87 cells, and downregulating NRP2 expression can significantly promote CHIKV infection.

[0014] Based on this, in a first aspect, the present invention provides a novel target for resisting chikungunya virus infection, namely neuropilin 2 (NRP2).

[0015] A second aspect of the invention provides the use of neuropilin 2 (NRP2) in the preparation of medicaments for the prevention or treatment of chikungunya virus infection.

[0016] Furthermore, the application refers to using neuropilin 2 (NRP2) as an intervention target for the prevention or treatment of chikungunya virus infection.

[0017] Furthermore, the diseases caused by the chikungunya virus infection include, but are not limited to, fever, joint pain, joint swelling, muscle pain, headache, nausea, fatigue, and rash. That is, the present invention also provides the use of neuropilin 2 (NRP2) in the preparation of medicaments for the prevention or treatment of diseases caused by chikungunya virus infection, including but not limited to fever, joint pain, joint swelling, muscle pain, headache, nausea, fatigue, and rash.

[0018] Furthermore, the drug is a drug that inhibits chikungunya virus infection by suppressing or downregulating the expression of neuropilin 2 (NRP2).

[0019] A third aspect of the invention provides the use of reagents that inhibit or downregulate the expression of neuropilin 2 (NRP2) in the preparation of medicaments for the prevention or treatment of chikungunya virus infection.

[0020] Furthermore, the reagents used to inhibit or downregulate the expression of NRP2 refer to siRNA, shRNA, miRNA, or antisense nucleotides that specifically interfere with the expression and processing of the NRP2 gene, or recombinant vectors (such as plasmids) containing siRNA, shRNA, miRNA, or antisense nucleotides.

[0021] In one embodiment of the present invention, the agent for inhibiting or downregulating the expression level of NRP2 is a neuropilin 2 interfering RNA (siRNA), the sequence of which is selected from any of the following:

[0022] CGACUGCAAGUAUGACUUUAUTT (SEQ ID NO: 1),

[0023] CCUCAACUUCAACCCUCACUUTT (SEQ ID NO:2),

[0024] GAUUGUCCUCAACUUCAACCC (SEQ ID NO: 3).

[0025] Among them, the siRNA shown in SEQ ID NO:3 showed the best effect in downregulating the expression of NRP2, but the siRNA shown in SEQ ID NO:1 showed the most significant reduction in CHIKV infection of SH-SY5Y cells.

[0026] In a fourth aspect, the present invention provides a medicament for the prevention or treatment of chikungunya virus infection, said medicament comprising a reagent that inhibits or downregulates the expression of NRP2.

[0027] The advantages of this invention are:

[0028] This invention has identified a novel host cell molecule, NRP2, that can inhibit CHIKV infection of SH-SY5Y and U87 cells. Downregulation of the NRP2 gene does not affect normal cellular physiological functions but significantly inhibits CHIKV infection of SH-SY5Y and U87 cells. Therefore, this invention provides a new target and treatment strategy for the clinical prevention and treatment of diseases caused by CHIKV infection, such as fever, joint pain, joint swelling, muscle pain, headache, nausea, fatigue, and rash. Attached Figure Description

[0029] Figure 1 To detect the effect of downregulation of various proteins on CHIKV infection using immunofluorescence assay, among which, Figure 1 A shows the fluorescence observation of viral infectivity in U87 cells after downregulation of various proteins. Figure 1 B is a fluorescence observation diagram of viral infectivity after downregulation of various proteins in SH-SY5Y cells;

[0030] CTRL: U87 / SH-SY5Y cells transfected with non-targeting siRNA (negative control group);

[0031] siRNA: U87 / SH-SY5Y cell groups (experimental group) transfected with siRNA targeting each target gene.

[0032] Figure 2 After transfecting different sequences of siRNA targeting NRP2, the mRNA level of the target gene and the CHIKV RNA level (expressed as relative expression level RQ value) were detected by real-time quantitative PCR (RT-PCR).

[0033] SiNC+V: SH-SY5Y cells transfected with non-targeting siRNA and infected with the virus (negative control group);

[0034] SiNRP2-1+V: SH-SY5Y cells transfected with siRNA (SEQ ID NO:1) targeting the NRP2 gene and infected with the virus;

[0035] SiNRP2-2+V: SH-SY5Y cells transfected with siRNA (SEQ ID NO:2) targeting the NRP2 gene and infected with the virus;

[0036] SiNRP2-3+V: SH-SY5Y cells transfected with siRNA (SEQ ID NO:3) targeting the NRP2 gene and infected with the virus;

[0037] +V: SH-SY5Y cells infected with the virus without transfecting any siRNA (simple infection group).

[0038] blank: SH-SY5Y cells that were not transfected with any siRNA and were not infected with the virus (blank control group).

[0039] Figure 3 To investigate the effects of downregulating NRP2 on target protein expression and CHIKV infection, the expression levels of NRP2 protein and viral protein were detected by Western blotting.

[0040] SiNC+V: SH-SY5Y cells transfected with non-targeting siRNA and infected with the virus (negative control group);

[0041] SiNRP2-1+V: SH-SY5Y cells transfected with siRNA (SEQ ID NO:1) targeting the NRP2 gene and infected with the virus;

[0042] SiNRP2-2+V: SH-SY5Y cells transfected with siRNA (SEQ ID NO:2) targeting the NRP2 gene and infected with the virus;

[0043] SiNRP2-3+V: SH-SY5Y cells transfected with siRNA (SEQ ID NO:3) targeting the NRP2 gene and infected with the virus;

[0044] +V: SH-SY5Y cells infected with the virus without transfecting any siRNA (simple infection group).

[0045] blank: SH-SY5Y cells that were not transfected with any siRNA and were not infected with the virus (blank control group). Detailed Implementation

[0046] The specific implementation methods provided by the present invention will be described in detail below with reference to the embodiments, but the implementation of the present invention is not limited thereto.

[0047] All reagents and raw materials used in this invention are commercially available or can be prepared according to literature methods. Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions as described in Sambrook et al., *Molecular Cloning: A Laboratory Guide* (New York: Cold Spring Harbor Laboratory Press, 1989), or under standard conditions, or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0048] Example 1:

[0049] 1. Design and synthesize specific siRNA sequences for each transport-related membrane protein.

[0050] 1.1 For each target gene, the full sequence and mRNA sequence were obtained by searching NCBI GeneBank. Biological analysis of AXIIR was performed using existing online resources and commonly used software to select coding regions as target sequences for siRNA design. Following siRNA design principles, the sequences were compared with human genome sequences using the BLAST function of the GeneBank database to ensure no homology. Potential siRNAs with eight consecutive bases at the 5' end of the aitisense strand paired with other genes were excluded; any potential siRNA with 14 consecutive bases paired with other genes was also excluded. Pre-evaluation was performed using design software, and three target sites with optimal kinetic parameters were selected for subsequent experimental procedures. Three interference sequences were synthesized for each gene, as shown in Table 1.

[0051] 1.2 The synthesis and purification of single-stranded siRNA were performed by Invitrogen.

[0052] Table 1. Design of siRNA targets

[0053] Gene NO. Target sequence information (5'-3') AXL 01 CGGACCACUGAAGCUACCUUGAACA AXL 02 CGGUGUCUGUUUCCAGGGUUCUGAA AXL 03 GACGUAUCGCCAAGAUGCCAGUCAA LIFR 01 UGACUUGCGACUACGUCAUUATT LIFR 02 ACUUCUGCAGAUUCGAUAUUATT LIFR 03 GUAGGCUCAGACAUAACAUUUTT PLXNB1 01 CAUGCUCUUUCGAGGGAUUAATT PLXNB1 02 CGACGUGCAAACAUCUGAUAATT PLXNB1 03 CUGCCACAUCCUAGGUCUAAGTT SLC3A2 01 CUAGCUCAUACCUGUCUGAUUTT SLC3A2 02 AGUCUCUUGCAAUCGGCUAAATT SLC3A2 03 GCCUACUCGAAUCCAACAAAGTT TGFBR3 01 CACAAGUCUGUUGUGUUCCUGCUCA TGFBR3 02 CAAGAACUGGGAUUUGCCAUCCAAA TGFBR3 03 CAGCUUCCUGUUUCUUCCCAUACUU ADGRB1 01 GCGGCACCAAGACAUGUUCCA ADGRB1 02 GGUGAUCUCCGUGACUGUGAA ADGRB1 03 GGCUCUGGACACGAGCUACGU CNTFR 01 CCAACACCUUCAAUGUGACUGUGCU CNTFR 02 CACAAUGCCACAGCUAUCACCUUUG CNTFR 03 CAGUCCCUUCUAGUCUGACUCAAUG ROBO3 01 CCCGGACGACAGAUAUUACAATT ROBO3 02 GCAUUGAGAAUAUCAUGAGUGTT ROBO3 03 GGCAGAGAGCUAGCUCCUCCC ADGRL3 01 GACUGUUCUGAGUUGAUAUAATT ADGRL3 02 AGCGUACAAUGACAGGUUAUUTT ADGRL3 03 CCGUGUCCAGGAACCUAUAAATT ADRA1B 01 GAGUCCCUCUCUACUUGUAUUTT ADRA1B 02 GGAUCCAUUCCAAGAACUUUCTT [[ID=6�]]ADRA1B 03 UCAUGUACUGCCGUGUCUAUATT CD44 01 GGACCAAUUACCAUAACUAUUTT CD44 02 CCGUUGGAAACAUAACCAUUATT CD44 03 CCUCCCAGUAUGACACAUAUUTT DPP4 01 UCAAUAUCUCCUGAUGGCAGUUUA DPP4 02 CAGUACUAUUCUGUGUCAUUCAGUA DPP4 03 CAGCAGAUGAUAACGUUCACUUCA GALR2 01 GCACUUCCGCAAAGGCUUCCG GALR2 02 GGUGACACGCAUGAUCCUCAU GALR2 03 GGGCGUGGACUUCCUCAUCUU GNB1 01 GCUUGUGAUGCUUCAGCCAAATT GNB1 02 GCCAUUUGCUUCUUUCCAAAUTT GNB1 03 CGAGCAACUUAAGAACCAGAUTT GP6 01 CAGACUCGGUAUGGCUUUGACCAAU GP6 02 CCUGCACACUGUGGAUGACAUGGUA GP6 03 GGCAGUUUGGGUAUGUCAUUCAUUU GRIK3 01 CGGAUCGGAGGAACUUCGAGUAUG GRIK3 02 CAAGGAGGCUCAAUGGGAAGGAUUA GRIK3 03 CCCAAAGAUGCAGCCGGGUAUUUGAA GRIN1 01 GUGCGACCUGGUGACGACUGG GRIN1 02 GCACAACUACGAGAGUGCGGC GRIN1 03 GCUUCUCAGCGCGCAUCCUGG GRIN2A 01 GAUGACCAACGCUUGGUUAUUTT GRIN2A 02 CCGUCAGCAUUCCUACGAUAATT GRIN2A 03 GACAAUUUCAGAAACGUCAAAUTT GRIN2C 01 GGCCUUCUUGGAGCCAUAUAGTT GRIN2C 02 GUGACCUCAGUAGCCUCAAAUTT GRIN2C 03 CCGCAGUAACUACCGUGACAUTT HTR1E 01 GAUUCUCUAUUACCGGAUUUATT HTR1E 02 AGAUGCCGAGAGCAUACUUAGTT HTR1E 03 AUUCUCUAUUACCGGAUUUACTT NR2C1 01 GAUGAAUGUAGCAACUAUAUUTT NR2C1 02 GCAGGUGUCAACCAGUUAUUUTT NR2C1 03 GCCAAUGUGGUUACAUCAUUATT CD81 01 CAACAUCAUCAGCAACCUCUUTT CD81 02 GAUCAUGAUCUUCGAGAUGAUTT CD81 03 CACAUGUAGGUGCGUGUAUGTT PROKR2 01 UUGAAACCACGGAUGAAUUAUTT PROKR2 02 GUAGCAGAAGCUAUUGGAGUU PROKR2 03 CCCAUCGGCUUACUUUGCAACAGAA PTPRU 01 CGAAGCCUGAGAUGGUCUAUGTT PTPRU 02 UGACGCGUUGCCACACCUAUATT PTPRU 03 GUACUGGGACUUGGCAUUUAGTT TYRO3 01 UUGGUAUCUCAGGUCUGAAUCTT TYRO3 02 CCAGUGACUGUCGGUACAUACTT TYRO3 03 CCGGUCCUUCAAUCGAGAAAGTT DAG1 01 CGAGUGACCAUUCCAACAGAUTT DAG1 02 CGGUGGUGAAUAACAGACUAUTT DAG1 03 CGUGGGCAAACACGAGUAUUUTT EGFR 01 UGUCUGGAAACAGUCCUGCUCCUCA EGFR 02 CAGUGGCAGAUUUGCAGACACAGUG EGFR 03 GAAACAGUGGCAGAUUUGCAGACAC FYN 01 GGGCUGUGUGCAAUGUAAGGAUAAA FYN 02 GAGGAGGAACAGGAGUGACACUCUU FYN 03 CAGGCAGAAGAGUGGUACUUUGGAA HSPB1 01 CCGAUGAGACUGCCGCCAAGUTT HSPB1 02 GAUCACCAUCCCAGUCACCUUTT HSPB1 03 CCCGGACGAGCUGACGGUCAATT ITGB4 01 GUGGAUGAGUUCCGGAAUAAATT ITGB4 02 GAGAAGCUUCACACCUAUUUCTT ITGB4 03 GAGGGUGUCAUCACCAUUGAATT LGR6 01 CGGCAUUCCAGUACCUGCCUAAACU LGR6 02 CAAGUUUGUGGUAGGUGCGAUUGCA LGR6 03 UAGCCUUCUCUGAUGUGGAUCUCAU NRP2 01 CGACUGCAAGUAUGACUUUAUTT NRP2 02 CCUCAACUUCAACCCUCACUUTT NRP2 03 GAUUGUCCUCAACUUCAACCC

[0054] 2siRNA transfection

[0055] The transfection procedure was performed according to the instructions for Lipofectamine™ 2000 (purchased from Invitrogen, catalog number 11668-019).

[0056] 1) U87 and SH-SY5Y cells were grown at a rate of 3 × 10⁻⁶. 4 The cells were seeded in 24-well cell culture plates and cultured at 37°C. When the cell density reached approximately 90%, siRNA transfection was performed.

[0057] 2) Add 1.5 μL of Lipofectamine™ 2000 to 50 μL of opti-MEM and mix gently. Incubate at room temperature for 5 minutes. Separately, mix 1 μL of 20 μM siRNA with 50 μL of opti-MEM. After incubation, add the diluted Lipofectamine™ 2000 transfection reagent to the diluted siRNA and mix gently by pipetting. After incubating at room temperature for 15 minutes, add the mixture to U87 and SH-SY5Y cells, and add 400 μL of opti-MEM to bring the final RNA concentration to 40 nM.

[0058] 3) Replace with DMEM medium (purchased from Thermo Fisher Scientific, catalog number 12430062) containing 10% fetal bovine serum (purchased from Thermo Fisher Scientific, catalog number 10437028) every 6-8 hours.

[0059] 3CHIKV virus infection of U87 and SH-SY5Y cells

[0060] 3.1 CHIKV virus infection experiment in U87 and SH-SY5Y cells

[0061] Forty-eight hours after transfection of U87 and SH-SY5Y cells with siRNA, CHIKV virus infection experiments were performed. The culture supernatant was aspirated, washed twice with pre-warmed PBS, and inoculated with CHIKV at an MOI of 0.5. After incubation at 37°C for 2 hours, the virus solution was discarded, and the cells were washed three times with pre-warmed PBS. Fresh culture medium was then added for further culture.

[0062] 3.2 Immunofluorescence staining to detect CHIKV antigen expression

[0063] U87 and SH-SY5Y cells were infected with the virus and cultured for another 24 hours. The expression of viral antigens was detected by immunofluorescence. The specific steps are as follows:

[0064] 1) Cell fixation and permeabilization: Remove the culture medium from the 96-well plate, wash the cells twice with PBS, add 100 μl of pre-cooled methanol (which has both fixation and permeabilization functions) to each well, fix and permeabilize at -20℃ for 20 min, and wash the cells three times with pre-cooled PBS.

[0065] 2) Sealing: Add 100 μl of 3% BSA to each well and incubate at room temperature for 2 h.

[0066] 3) Primary antibody incubation: Add 100 μl of CHIKV-specific rabbit monoclonal antibody (1:500 dilution) to each well, incubate overnight at 4°C on a shaker, and wash 3 times with pre-cooled PBS.

[0067] 4) Secondary antibody incubation: Add 100 μl of AF 488 fluorescently labeled anti-rabbit IgG (1:1000 dilution) to each well, incubate at room temperature in the dark for 2 h, and wash 3 times with pre-cooled PBS in the dark.

[0068] 5) Labeling cell nuclei: Add the nuclear fluorescent dye DAPI (1:10000, diluted with PBS) to each well, incubate at room temperature in the dark for 10 min, and wash 3 times with pre-cooled PBS in the dark.

[0069] 6) The percentage of green AF 488 positive cells was detected and calculated under a fluorescence microscope.

[0070] 4. Identification of the interference effect of SH-SY5Y cells and its impact on viral infection

[0071] 4.1 Detection of target protein mRNA levels using real-time quantitative PCR (RT-PCR)

[0072] 1) Extract total RNA from each group of cells. The specific steps are as follows:

[0073] Forty-eight hours after transfection, discard the culture supernatant. Add 0.5 ml of RNAiso Plus (purchased from TAKARA, catalog number 9109) to the cells and mix thoroughly. Lyse the cells on ice for 5-10 minutes. Transfer the mixture to an EP tube, add 1 / 5 volume of chloroform, and invert to mix 8 times. Centrifuge at 12,000 rpm for 15 minutes at 4°C. Transfer the upper aqueous phase to a new EP tube, add an equal volume of isopropanol, mix thoroughly, and incubate at room temperature for 10 minutes. Centrifuge at 12,000 rpm for 15 minutes at 4°C. Discard the supernatant and add 0.8 ml of ice-cold 75% ethanol. Centrifuge at 12,000 rpm for 10 minutes at 4°C. Discard the supernatant completely, air-dry the RNA precipitate at room temperature, and dissolve the precipitate in RNase-free dH2O to obtain total RNA.

[0074] 2) Total cDNA was prepared using a reverse transcription kit (purchased from TAKARA, catalog number RR036A). The specific steps are as follows:

[0075] Add the following reaction mixture to the PCR tube.

[0076] 5×PrimeScript RT Master Mix 4 μL

[0077] Total RNA 1000 ng

[0078] Rnase-free dH2O up to 20 μL

[0079] Mix gently and incubate at 37°C for 30 minutes (reverse transcription reaction).

[0080] 3) RT-PCR detection of target gene expression levels

[0081] The reaction was carried out using the TB Green Premix Ex Taq kit (purchased from TAKARA, catalog number RR420A). The reaction system is as follows:

[0082] SYBR Premix Ex Taq 10 μL

[0083] Forward Primer (10μM) 1 μL

[0084] Revers Primer (10μM) 1 μL

[0085] Rox 0.4 μL

[0086] 2 μL DNA template

[0087] dH2O 5.6 μL

[0088] Total 20 μL

[0089] Two-step amplification was performed using an Applied Biosystems 7300 plus instrument:

[0090] Step 1: Pre-denaturation at 95℃ for 30 seconds,

[0091] Step 2: 95℃ for 5 seconds

[0092] 60℃ for 30 seconds

[0093] 4) The relative expression levels of each target gene were calculated using the ΔΔt method.

[0094] RQ=2 -ΔΔt =2 -[(Ct处理样本目的基因-Ct对照样本目的基因)- (Ct处理样本内参基因-Ct对照样本内参基因)]

[0095] Interference efficiency = 1 - RQ

[0096] 4.2 Western blot of proteins.

[0097] (1) Total protein was extracted from each group of SH-SY5Y cells using protein lysis buffer RIPA.

[0098] (2) After protein quantification, 20 μg of protein was added to a 12.5% ​​SDA-PAGE gel for electrophoresis, and the corresponding bands were transferred to a PVDF membrane using an electroporator.

[0099] (3) Non-specific sites of the protein were blocked with 5% skim milk, and then incubated overnight at 4°C with diluted CHIKV-specific rabbit polyclonal antibody CHIKV E1 (1:5000 dilution) or diluted NRP2-specific rabbit polyclonal antibody (1:1000 dilution), and washed three times with TBST buffer.

[0100] (4) Then incubate with HRP-labeled goat anti-rabbit IgG (1:10000 dilution) at room temperature for 2 hours, and then wash three times with TBST buffer.

[0101] (5) Finally, develop the color using the colorimetric solution and take a picture for analysis.

[0102] Experimental results:

[0103] 1. Design, synthesize, and screen effective siRNAs

[0104] For each target gene sequence, we designed multiple RNA interference target sequences and used design software for pre-evaluation. We selected three optimal kinetic parameter targets for subsequent experimental procedures. A total of three interference sequences were synthesized for each gene, as shown in Table 1.

[0105] Interference RNA for various genes was transfected into U87 / SH-SY5Y cells using in vitro transfection. After 48 hours, the cells were infected with CHIKV for 24 hours. Immunofluorescence results showed that downregulation of the NRP2 gene significantly reduced the inhibitory effect of CHIKV on U87 cells. Figure 1 A) and SH-SY5Y cells ( Figure 1 Infection with B) was ultimately chosen, and NRP2, which had the greatest impact on CHIKV infection, was selected for subsequent experiments. Figure 1 ).

[0106] Effects of 2siRNA interference on CHIKV virus infection

[0107] To further clarify the important role of NRP2 in CHIKV infection, the effects of transfecting three siRNAs targeting NRP2 on viral infectivity were observed. The interference efficiency of the siRNAs was detected by RT-PCR and Western blotting. CHIKV viral load was then detected by RT-PCR and Western blotting, respectively. The results showed that different siRNAs had varying interference efficiencies towards NRP2. Figure 2B, 3), among which NRP2 siRNA (SEQ ID NO:3) had the highest interference efficiency. The effect of interference on CHIKV infectivity was examined, and it was found that all three NRP2 siRNAs showed an inhibition rate of over 50% against viral infection, but NRP2 siRNA (SEQ ID NO:1) had the greatest impact on CHIKV infection. Figure 2 A, 3). These results indicate that NRP2 molecules play an important role in CHIKV infection of SH-SY5Y cells.

[0108] Therefore, NRP2 can serve as a novel host target for inhibiting CHIKV infection of U87 and SH-SY5Y cells.

[0109] The above experimental results demonstrate that this invention has identified a novel host cell molecule, NRP2, that can influence CHIKV infection of SH-SY5Y cells. Downregulation of the NRP2 gene significantly inhibits CHIKV infection of U87 and SH-SY5Y cells. Therefore, this invention provides a new target and treatment strategy for the clinical prevention and treatment of CHIKV infection.

[0110] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. Application of neurocilia protein 2 in the preparation of drugs for the prevention or treatment of chikungunya virus infection.

2. The use of the neurocilia protein 2 according to claim 1 in the preparation of a medicament for the prevention or treatment of chikungunya virus infection, characterized in that, The use of the described neurocilia protein 2 in the preparation of medicaments for the prevention or treatment of fever, arthritis, arthralgia, joint swelling, muscle pain, headache, nausea, fatigue and rash caused by Chikungunya virus infection.

3. The use of the neurocilia protein 2 according to claim 1 in the preparation of a medicament for the prevention or treatment of chikungunya virus infection, characterized in that, The drug described is a drug that inhibits chikungunya virus infection by suppressing or downregulating the expression of neurocilia 2.

4. Application of reagents that inhibit or downregulate the expression of neurocilia 2 in the preparation of drugs for the prevention or treatment of chikungunya virus infection.

5. The use of the reagent according to claim 4 that inhibits or downregulates the expression of neurocilia protein 2 in the preparation of drugs for the prevention or treatment of chikungunya virus infection, characterized in that, The reagents that inhibit or downregulate the expression of neurocilia 2 refer to siRNA, shRNA, miRNA, or antisense nucleotides that specifically interfere with the expression and processing of the neurocilia 2 gene, or recombinant vectors containing siRNA, shRNA, miRNA, or antisense nucleotides.

6. The use of the reagent according to claim 4 that inhibits or downregulates the expression of neurocilia protein 2 in the preparation of drugs for the prevention or treatment of chikungunya virus infection, characterized in that, The reagent used to inhibit or downregulate the expression of neurocilia 2 is an interfering RNA of neurocilia 2, and the nucleotide sequence of the interfering RNA is shown in any one of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:

3.

7. A drug for the prevention or treatment of chikungunya virus infection, characterized in that, The drug contains a reagent that inhibits or downregulates the expression of neurofibrillary protein 2.

Images