Use of gosr1 gene in prevention and / or treatment of respiratory pathogens

Abstract

The application relates to the field of biological medicine, and discloses application of a GOSR1 gene in prevention and / or treatment of respiratory pathogenic bacteria. By specifically knocking down the GOSR1 gene, the application can reduce the expression level of pathogenic gene and protein, thereby significantly inhibiting the proliferation of the respiratory pathogenic bacteria. In addition, it is disclosed that overexpression of the GOSR1 gene can activate the proliferation of the pathogenic bacteria in cells infected by the respiratory pathogenic bacteria. The application provides a brand-new strategy for treatment of respiratory pathogenic bacteria infection based on the regulation of the GOSR1 gene on the proliferation of the respiratory pathogenic bacteria, and is especially suitable for intervention of influenza A virus infection.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of the GOSR1 gene in the prevention and / or treatment of respiratory pathogens. Background Technology

[0002] Highly transmissible influenza viruses refer to a class of influenza virus strains that can primarily spread via aerosols and possess stronger airborne transmission capabilities. A typical example is influenza A virus. Due to the ease with which its genome can recombine and mutate, influenza A virus is a major source of strains with high aerosol transmission capabilities. The surface antigens of influenza A viruses (hemagglutinin HA and neuraminidase NA) are highly susceptible to mutation. When mutation sites affect the binding ability of viral particles to aerosol droplets and their stability in the air, the strain can evolve to have a stronger aerosol transmission potential. For example, some influenza A H1N1 and H3N2 subtypes, through amino acid mutations in the stem region of the HA protein, reduce the inactivation rate of the virus in dry air, allowing it to attach to aerosol particles with a diameter <5 μm, remain suspended in enclosed spaces for several hours, and achieve long-distance transmission.

[0003] Compared to influenza B and C viruses, influenza A viruses not only have a wider host range (infecting humans, poultry, pigs, and other animals), but also have a higher frequency of gene recombination, making them more likely to produce novel strains with both high pathogenicity and high aerosol transmissibility. Therefore, they have become the core subject of influenza aerosol transmission research and a key focus of global influenza surveillance and control systems.

[0004] Currently, research on inhibiting the proliferation and spread of influenza A virus is limited by the virus's high variability and drug resistance, coupled with difficulties in finding drug targets, poor broad-spectrum and timeliness of vaccines, and significant bottlenecks in aerosol transmission blocking technology. There is an urgent need to explore new technical paths and research directions. Summary of the Invention

[0005] The purpose of this invention is to overcome the aforementioned problems in the prior art and to provide the application of the GOSR1 gene in the prevention and / or treatment of respiratory pathogens.

[0006] To achieve the above objectives, the first aspect of the present invention provides the use of an agent that knocks down GOSR1 gene expression in the preparation of a medicament for the prevention and / or treatment of respiratory pathogen infections.

[0007] A second aspect of the present invention provides the use of a formulation that knocks down GOSR1 gene expression in the preparation of a medicament for inhibiting the proliferation of respiratory pathogens.

[0008] A third aspect of the present invention provides the use of a formulation that knocks down GOSR1 gene expression in the preparation of a medicament for reducing the expression of respiratory pathogen proteins.

[0009] The fourth aspect of this invention provides the use of a formulation that knocks down the expression of the GOSR1 gene in the in vitro inhibition of pathogen proliferation in cells infected with respiratory pathogens.

[0010] The fifth aspect of this invention provides the use of a formulation that knocks down the expression of the GOSR1 gene in reducing the production of pathogen proteins in cells in vitro to reduce respiratory pathogen infection.

[0011] The sixth aspect of this invention provides the use of a formulation overexpressing the GOSR1 gene in enhancing pathogen proliferation in cells infected with respiratory pathogens in vitro.

[0012] Through the above technical solutions, this invention proposes for the first time that specifically knocking down the GOSR1 gene can significantly inhibit the proliferation of respiratory pathogens and reduce their protein expression levels. Furthermore, this invention also demonstrates that overexpression of the GOSR1 gene can activate pathogen proliferation in cells infected with respiratory pathogens. Based on the regulatory role of the GOSR1 gene in the proliferation of respiratory pathogens, this invention provides a novel targeted intervention strategy for the treatment of respiratory pathogen infections, particularly suitable for the prevention and control of influenza A virus infection.

[0013] Biological Preservation

[0014] The cells provided in this invention are immortalized guinea pig tracheal epithelial cells, deposited on December 3, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 46763. The immortalized guinea pig tracheal epithelial cells provided in this invention are named GTE1.

[0015] The cells provided in this invention are immortalized guinea pig tracheal fibroblast cells, deposited on December 3, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 46764. The immortalized guinea pig tracheal fibroblast cells provided in this invention are named GTF1. Attached Figure Description

[0016] Figure 1A This involves Western blot (WB) to detect the expression level of viral nucleoprotein (NP) in tracheal epithelial cells with GOSR1 knocked down by siRNA after infection with H1 or H5 subtype influenza virus. p<0.05, p<0.0001);

[0017] Figure 1B and Figure 1C It is a quantitative analysis of viral M gene mRNA levels using qPCR (Quantitative Real-time polymerase chain reaction). p<0.05);

[0018] Figure 1D The purpose is to observe the localization and infection rate of viral matrix protein (M) in tracheal epithelial cells using immunofluorescence.

[0019] Figure 2A Western blot analysis was performed to detect the expression level of viral NP protein in tracheal fibroblasts with GOSR1 knocked down by siRNA after infection with H1 or H5 subtype influenza virus. p<0.001, p<0.0001);

[0020] Figure 2B and Figure 2C It is a qPCR quantitative analysis of viral M gene mRNA levels ( p<0.05, p<0.001);

[0021] Figure 2D The immunofluorescence assay is used to observe the localization and infection rate of the viral M protein in tracheal fibroblasts.

[0022] Figure 3A Western blot analysis was performed to detect the expression level of viral NP protein in tracheal epithelial cells overexpressing GOSR1 after infection with H1 or H5 subtype influenza virus. p<0.001, p<0.0001);

[0023] Figure 3B and Figure 3C It is a real-time quantitative PCR method for quantitative analysis of viral M gene mRNA levels. p<0.01, p<0.001);

[0024] Figure 3D The immunofluorescence assay is used to observe the localization and infection rate of the viral M protein in tracheal epithelial cells.

[0025] Figure 4A Western blot analysis was used to detect the expression level of viral NP protein in tracheal fibroblasts overexpressing GOSR1 after infection with H1 or H5 subtype influenza virus. p<0.001);

[0026] Figure 4B and Figure 4C It is a qPCR quantitative analysis of viral M gene mRNA levels ( p<0.05, p<0.01);

[0027] Figure 4D The immunofluorescence assay is used to observe the localization and infection rate of the viral M protein in tracheal fibroblasts.

[0028] Figure 5 This is a schematic diagram of an experimental method for detecting the aerosol transmission ability of viruses among guinea pigs.

[0029] Figure 6A The results show the viral titer measurements of the H1 virus inoculation group and the aerosol transmission group in the negative control group at different testing days.

[0030] Figure 6B The results show the viral titer determination of the H1 virus inoculation group and the aerosol transmission group in the GOSR1 group at different test days.

[0031] Figure 6C These are the results of serum antibody testing in guinea pigs in the negative control group and the GOSR1 knockdown group;

[0032] Figure 6D The results show the viral titer of H1 in the nasal turbinates, trachea, and lung tissue of guinea pigs in the negative control group.

[0033] Figure 6E The results show the viral titer of H1 in the nasal turbinates, trachea, and lung tissue of guinea pigs with knocked-down GOSR1 group.

[0034] Figure 6F These are the results of virus concentration measurements in exhaled aerosols of guinea pigs infected with H1 in the negative control group and the GOSR1 knockdown group. Detailed Implementation

[0035] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0036] In this invention, it is understood that knocking down GOSR1 gene expression includes reducing the transcription or translation of the GOSR1 gene through technical means (such as RNAi, CRISPR, etc.), thereby reducing the amount of its mRNA or protein synthesis, as well as affecting its function at the protein level, which may be done by inhibiting its enzyme activity, changing its conformation, or blocking its interaction with other molecules.

[0037] In this invention, the GOSR1 gene (Golgi SNAP receptor complex member 1 Gene) encodes a transport membrane protein that is a core component of the Golgi SNAP receptor (soluble n-ethylmaleimide-sensitive fusion protein attachment protein receptor, SNARE) complex, primarily involved in protein transport between the endoplasmic reticulum and the Golgi apparatus, as well as within the Golgi apparatus. This protein plays an upstream or internal role in vesicle-mediated transport, mediating anterograde and retrograde transport through vesicle fusion. The GOSR1 gene (human) has a Gene ID of 9527 on the NCBI website, the GOSR1 gene (guinea pig) has a Gene ID of 100716724 on the NCBI website, the GOSR1 gene (mouse) has a Gene ID of 53334 on the NCBI website, and the GOSR1 gene (rat) has a Gene ID of 94189 on the NCBI website. GOSR1 is also known as P28, GS28, GOS28, GOLIM2, GOS-28, and GOS28 / P28.

[0038] In this invention, the GOSR1 gene can be the guinea pig GOSR1 gene, and the specific nucleic acid sequence is shown in SEQ ID NO:3.

[0039] 5'--3' (SEQ ID NO: 3).

[0040] The first aspect of the present invention provides the use of a formulation that knocks down GOSR1 gene expression in the preparation of a medicament for the prevention and / or treatment of respiratory pathogen infections.

[0041] A second aspect of the present invention provides the use of a formulation that knocks down GOSR1 gene expression in the preparation of a medicament for inhibiting the proliferation of respiratory pathogens.

[0042] A third aspect of the present invention provides the use of a formulation that knocks down GOSR1 gene expression in the preparation of a medicament for reducing the expression of respiratory pathogen proteins.

[0043] The fourth aspect of this invention provides the use of a formulation that knocks down the expression of the GOSR1 gene in inhibiting pathogen proliferation in cells infected with respiratory pathogens (in vitro).

[0044] The fifth aspect of the present invention provides the use of a formulation that knocks down the expression of the GOSR1 gene in reducing the production of pathogen proteins in cells (in vitro) to reduce respiratory pathogen infection.

[0045] In this invention, preferably, the formulation comprises a nucleic acid drug. In this invention, the formulation for knocking down GOSR1 gene expression can be selected from any nucleic acid molecule or composition that can specifically target the mRNA of the GOSR1 gene and downregulate its expression level, including but not limited to small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), antisense oligonucleotide (ASO), or guide RNA (gRNA) compositions of the CRISPR-Cas9 gene editing system that target the GOSR1 gene.

[0046] In this invention, the siRNA is a double-stranded RNA molecule with a length of 19-25 nucleotides (preferably 19-23 nucleotides), consisting of a sense strand and an antisense strand, which form a double-stranded structure through complementary base pairing. When the siRNA is introduced into a cell, it binds to intracellular proteins such as Argonaute to form an RNA-induced silencing complex (RISC). Subsequently, the sense strand in the RISC is degraded, while the antisense strand specifically recognizes and binds to the target mRNA (messenger RNA) whose sequence is complementary to its own. Finally, the RISC cleaves the target mRNA through endonuclease activity or inhibits the translation process of the target mRNA, thereby blocking the expression of the target gene.

[0047] In this invention, only one set of siRNAs targeting the GOSR1 gene is provided as an example. To comprehensively cover the target gene's action site and improve the stability and reliability of gene silencing effects, a series of siRNAs can be specifically designed to completely cover the entire sequence of the GOSR1 gene. By targeting different regions of the gene, efficient inhibition of GOSR1 gene expression can be achieved, avoiding the problem of inhibition failure caused by mutations at a single target site. Furthermore, this invention can design corresponding series of siRNAs based on the nucleotide sequence characteristics of the GOSR1 gene in different species, and the siRNAs corresponding to each species can completely cover the entire sequence of its own GOSR1 gene, ensuring specific targeting effects on the GOSR1 gene in different species.

[0048] In this invention, preferably, the nucleic acid drug is selected from siRNA, the sense strand sequence of which is shown in SEQ ID NO: 1 and the antisense strand sequence is shown in SEQ ID NO: 2.

[0049] In this invention, preferably, the formulation further includes a delivery carrier; the delivery carrier may be selected from one or more of liposomes, cationic polymers, viral vectors or cell-penetrating peptides, which can form a stable complex with the active ingredient that knocks down GOSR1 gene expression, protecting the active ingredient from degradation by nucleases, while enhancing the targeted delivery capability of the formulation.

[0050] More preferably, the delivery carrier is a liposome.

[0051] This invention also provides the application of a formulation that knocks down GOSR1 gene expression in the in vitro inhibition of pathogen proliferation in cells infected with respiratory pathogens.

[0052] In this invention, preferably, the cells are tracheal epithelial cells and / or tracheal fibroblasts.

[0053] In this invention, preferably, the pathogen protein includes at least one of nucleoprotein (NP), hemagglutinin (HA), neuraminidase (NA), RNA polymerase basic proteins (PB1, PB2 and PA), matrix protein (M), and non-structural high protein (NS).

[0054] The sixth aspect of this invention provides the use of a formulation overexpressing the GOSR1 gene in (in vitro) enhancing pathogen proliferation in cells infected with respiratory pathogens.

[0055] In this invention, preferably, the cells are tracheal epithelial cells and / or tracheal fibroblasts, more preferably tracheal fibroblasts.

[0056] In this invention, preferably, the preparation for overexpressing the GOSR1 gene includes a recombinant plasmid that overexpresses the GOSR1 gene, which, after transfection of host cells, can significantly upregulate the mRNA and protein expression levels of the GOSR1 gene.

[0057] More preferably, the GOSR1 gene overexpression insert sequence in the recombinant plasmid is shown in SEQ ID NO: 10.

[0058] More preferably, the negative control is an empty pCDNA3.1 vector plasmid without any other sequences inserted.

[0059] In this invention, preferably, the respiratory pathogen is at least one of influenza virus, novel coronavirus (SARS-CoV-2), respiratory syncytial virus, rhinovirus, parainfluenza virus and adenovirus.

[0060] More preferably, the respiratory pathogen is an influenza virus; more preferably, it is influenza A virus H1 and / or influenza A virus H5.

[0061] The present invention will be described in detail below through embodiments. It should be understood that the following embodiments are only used to further explain and illustrate the content of the present invention by way of example, and are not intended to limit the present invention.

[0062] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available products purchased from regular chemical or biological reagent / material suppliers, and all reagents are of analytical grade.

[0063] The tracheal epithelial cells were immortalized guinea pig tracheal epithelial cells, provided by the Academy of Military Medical Sciences of the Chinese People's Liberation Army, with accession number CGMCC No. 46763;

[0064] The tracheal fibroblasts were immortalized tracheal fibroblasts from guinea pigs, provided by the Academy of Military Medical Sciences of the Chinese People's Liberation Army, with accession number CGMCC No. 46764;

[0065] The influenza virus H1 was provided by the Academy of Military Medical Sciences of the Chinese People's Liberation Army, with the strain number A / California / 04 / 2009 (H1N1); the Genbank accession number for H1N1 is GCA_038510525.1;

[0066] The influenza virus H5 was provided by the Academy of Military Medical Sciences of the Chinese People's Liberation Army, with the strain number A / chicken / Hebei / 3399 / 2017(H5N6); the HA and NA gene sequences of H5N6 correspond to Genbank accession numbers PX884831 and PX884830, respectively.

[0067] Example 1

[0068] This example illustrates that knocking down the GOSR1 gene can inhibit the production of influenza virus in tracheal epithelial cells and tracheal fibroblasts.

[0069] 1. Design small interfering RNA (siRNA) targeting the GOSR1 gene.

[0070] Table 1

[0071]

[0072] The nucleic acid sequences in Table 1 were all synthesized by Sangon Biotech (Shanghai) Co., Ltd. dT stands for deoxythymidine nucleotide modification. The control group RNA is a meaningless RNA sequence.

[0073] 2. Cell transfection and viral infection

[0074] (1) Cell transfection

[0075] Tracheal epithelial cells or tracheal fibroblasts in the logarithmic growth phase were digested with trypsin, centrifuged, resuspended, and counted at 5 × 10⁶ cells per well. 4 Cells were seeded into 24-well plates and cultured for 12 h. For transfection, the transfection reagent was prepared according to the Lipofectamine 3000 (Invitrogen, L3000015) kit. 50 nM siRNA and 1.5 μL Lipofectamine 3000 were added to each well. The plates were then incubated at 37°C in a 5% CO2 incubator for 6 h. The medium was then replaced with the above-mentioned epithelial medium or fibroblast medium containing 5% FBS (fetal bovine serum, GIBCO, 10099141C) and cultured for another 48 h.

[0076] (2) Viral infection

[0077] For H1 or H5 influenza virus infection experiments, the cell supernatant was discarded, and 100 μL of virus diluted with serum-free OPTI MEM medium (GIBCO, 31985070) (MOI=0.01) was added to each well of a 24-well plate. The plate was incubated at 37°C for 1 hour. After incubation, the viral supernatant was discarded, and 500 μL of maintenance medium (0.5% FBS + the above-mentioned epithelial or fibroblast medium) was added to each well. The plate was then returned to the incubator for another 24 hours.

[0078] 3. Measurement of viral protein expression levels and viral proliferation levels

[0079] (1) Western blot determination of viral protein expression levels: After viral infection of cells, cellular proteins were extracted and detected by Western blot using viral protein-specific antibodies. The band signals were then quantitatively analyzed to assess the relative expression levels of viral proteins. The specific steps are as follows:

[0080] a. After infection, discard the supernatant from each well of the cells, add 200 μL of prepared cell protein lysis buffer (Beyotime, P0013C and P1065), incubate on ice for 10 min, then collect the supernatant into a 1.5 mL centrifuge tube, centrifuge at 12000 rpm for 10 min at 4 °C, and transfer the supernatant to a new pre-chilled 1.5 mL centrifuge tube; add 5×SDS-PAGE loading buffer (Yisheng, 20315ES05) to the supernatant, boil at 100 °C for 10 min to denature the virus particles, ensuring protein release. Centrifuge at 12000 rpm for 1 min, and use the supernatant for Western blotting.

[0081] b. Place a 10% high-resolution precast gel (Yaxin, LK403) in an electrophoresis tank and add an appropriate amount of protein electrophoresis buffer. Add markers and protein samples to the gel wells, adjust the voltage to 80 V, and start electrophoresis. After 20 min, observe the lanes (running under the stacking gel). Once the bands have separated, adjust the voltage to 120 V and stop electrophoresis after 70 min. Cut a PVDF membrane (Millipore, IPVH00010) according to the gel size. Immerse the PVDF membrane in 100% methanol (Shanghai Test, 10010018) for 5 min and in 20% methanol for 2 min. Start the PVDF membrane transfer at a constant current of 400 mA for 30 min. Place the PVDF membrane in a container and block it with TBST (Yaxin, PS103S) containing 5% skim milk powder (Solepro, D8340) at room temperature for 1 h. After blocking, wash the membrane three times with TBST for 10 min each time.

[0082] c. Primary antibody: Place the PVDF membrane containing the target protein in a solution of influenza A virus nucleoprotein antibody (Xinbosheng, GTX636282-S) diluted 1:5000 using universal antibody dilution buffer. Place the PVDF membrane containing the internal control in a solution of actin antibody (MBL, M177-3) diluted 1:8000 using universal antibody dilution buffer. Incubate at 4°C on a shaker for 18 h. Wash the membrane three times with TBST for 10 min each time.

[0083] d. Secondary antibody: The PVDF membrane containing the target protein was placed in a solution of goat anti-rabbit antibody (Xinbosheng, GTX213110-01) diluted 1:5000 using a universal antibody diluent. The PVDF membrane containing the internal control was placed in a solution of goat anti-mouse antibody (Immunoway, RS0001) diluted 1:14000 using a universal antibody diluent. The membranes were incubated at room temperature for 1.5 h. The membranes were washed three times with TBST for 10 min each time. A developing solution (Pulley, P1010) was prepared to develop the PVDF membranes.

[0084] (2) qPCR determination of viral mRNA level: After viral infection of cells, cellular RNA was extracted, and RT-qPCR was performed using specific primers targeting viral M gene mRNA. The relative expression level of viral M gene mRNA was calculated using a relative quantification method. Specifically, the target gene and internal reference gene were detected in each sample. ① The ΔCt value of the target gene and internal reference gene was calculated: ΔCt = Ct (target gene) - Ct (internal reference gene). ② The ΔΔCt value between the treatment group and the control group was calculated: ΔΔCt = ΔCt (treatment group) - ΔCt (control group). ③ The relative expression level was calculated: 2^(-ΔΔCt). The specific steps are as follows:

[0085] a. After discarding the supernatant from each well of the infected cells, extract RNA using an RNA extraction kit (TransGold, ER111) according to the kit instructions. Measure the RNA concentration using NanoDrop and store at -80°C.

[0086] b. Using a reverse transcription kit (TAKARA, RR047), follow the kit instructions to reverse transcribe the RNA extracted in step a to obtain cDNA.

[0087] c. qPCR reaction: A qPCR detection kit (TransGold, AQ621) was used, and the qPCR reaction was performed according to the kit instructions. The upstream primer sequence for the M gene was 5'-CTTCTAACCGAGGTCGAAACG-3' (SEQ ID NO: 6), and the downstream primer sequence for the M gene was 5'-CTTTAGCCACTCCATGAGAGC-3' (SEQ ID NO: 7). The upstream primer sequence for the internal reference gene (GAPDH) was 5'-AACTTCGGCATTGTGGAGGG-3' (SEQ ID NO: 8), and the downstream primer sequence for the internal reference gene was 5'-GGATGCGGGGATGATGTTCT-3' (SEQ ID NO: 9). The qPCR reaction was performed according to the reaction conditions in Table 2.

[0088] Table 2

[0089]

[0090] Note: Fluorescence signal collection was performed in the second program segment (56℃ annealing and extension).

[0091] (3) Immunofluorescence experiment: After the virus infects the cells, the cells are fixed with formaldehyde, and the viral proliferation level is determined using virus protein-specific antibodies and immunofluorescence technology. The specific steps are as follows:

[0092] After discarding the supernatant from each well, add 500 μL of pre-chilled 4% paraformaldehyde fixative (Solepro, P1110) and incubate at room temperature for 30 min. After fixation, discard the 4% paraformaldehyde and wash three times with PBS for 5 min each time. Add 1 mL of 1% Triton X-100 solution to the washed cells and incubate at room temperature for 30 min. After permeation, wash three times with PBS for 5 min each time. After washing, add 1 mL of 3% BSA and incubate at 37°C for 30 min. After incubation, wash three times with PBS for 5 min each time. Add 200 µL of primary antibody (Xinbosheng, GTX636675, 1:200 dilution) and incubate overnight at 4°C. Wash three times with PBST on a shaker for 5 min each time. Then add 200 µL of secondary antibody (Polyenergy, AF594, 1:200 dilution) and incubate at 37°C in the dark for 2 h. Wash five times with PBST on a shaker for 5 min each time. Add 20 µL of DAPI staining solution (Solepro, S2110) to the washed cells and incubate at room temperature for 10 min.

[0093] Results of tracheal epithelial cells as follows Figure 1A As shown in Figure -D, the Western blot results indicated that in tracheal epithelial cells, after knocking down the GOSR1 gene via siRNA transfection, the relative expression level of NP protein in group H1 decreased from 0.93 to 0.18, and the relative expression level of NP protein in group H5 decreased from 0.68 to 0.24 (the average value of the NP bands normalized using β-actin as an internal control, the same below). The relative expression level of NP protein in group H1 was significantly lower than that in group H5. p<0.05) Figure 1A qPCR results showed that after knocking down GOSR1 in tracheal epithelial cells, the relative expression level of M gene mRNA in group H1 significantly decreased from 1 to 0.10 (…). p<0.05) Figure 1B The relative expression level of M gene mRNA in group H5 decreased significantly from 1 to 0.22. p<0.05) Figure 1C Immunofluorescence results showed that knocking down GOSR1 significantly reduced the proliferation levels of H1 and H5 in tracheal epithelial cells. Figure 1D ).

[0094] Tracheal fibrosis results as follows Figure 2A -D, after knocking down the GOSR1 gene in tracheal fibroblasts via siRNA transfection, the relative expression level of NP protein in group H1 decreased from 0.55 to 0.06, and the relative expression level of NP protein in group H5 decreased from 0.73 to 0.54. The relative expression level of NP protein in group H1 was significantly lower than that in group H5. p<0.0001) Figure 2A qPCR results showed that after knocking down GOSR1 in tracheal fibroblasts, the relative expression level of M gene mRNA in group H1 significantly decreased from 1 to 0.03. p<0.001) Figure 2B The relative expression level of M gene mRNA in group H5 decreased significantly from 1 to 0.49. p<0.05) Figure 2C Immunofluorescence results showed that knockdown of GOSR1 significantly reduced the proliferation levels of H1 and H5 in tracheal fibroblasts, with H1 proliferation being significantly lower than that of H5. Figure 2D ).

[0095] The above experiments show that knocking down the GOSR1 gene specifically with siRNA can inhibit the proliferation of influenza virus in tracheal epithelial cells and fibroblasts, thus indicating that knocking down the GOSR1 gene can be used to prevent and / or treat influenza virus infection.

[0096] Example 2

[0097] This example illustrates that overexpression of the GOSR1 gene can activate the production of influenza virus in tracheal epithelial cells and tracheal fibroblasts.

[0098] Design of overexpression recombinant plasmid: The insert sequence of the guinea pig GOSR1 overexpression plasmid is shown in SEQ ID No: 10.

[0099] 5'--3' (SEQ ID No: 10). The preparation steps of vector construction, transformation screening and identification of recombinant plasmids can all be completed by mature molecular biology methods in this field, and will not be described in detail here.

[0100] Tracheal epithelial cells or tracheal fibroblasts in the logarithmic growth phase were digested with trypsin, centrifuged, resuspended, and counted at 5 × 10⁶ cells per well. 4Cells were seeded in 24-well plates and cultured for 12 h. For transfection, the transfection reagent was prepared according to the Lipofectamine 3000 kit, with 500 ng of recombinant plasmid, 1.5 μL of Lipofectamine 3000, and 1 μL of P3000 added to each well. The plates were then incubated at 37°C in a 5% CO2 incubator for 6 h. The culture medium was then replaced with either epithelial culture medium containing 5% FBS (including DMEM / F12 (GIBCO, 21331020), 1% epithelial cell culture additive (Zhejiang Meisen Cell Technology Co., Ltd., CTCC-009-613-2), and 1% penicillin / streptomycin (GIBCO, 15140148)) or fibroblast culture medium (including DMEM / F12, 10 ng / mL). bFGF (Invitrogen, 13256-029), 1% Glutamax (GIBCO, 31765035), and 1% penicillin / streptomycin were cultured for 48 hours to construct tracheal epithelial cells or tracheal fibroblasts overexpressing the GOSR1 gene. Subsequently, H1 or H5 influenza virus infection experiments were performed (MOI=0.0001), following the virus infection steps in Example 1.

[0101] Western blot (WB) and qPCR experiments were used to detect the expression of the GOSR1 gene in each group, as well as the expression level of viral proteins in tracheal epithelial cells and tracheal fibroblasts that overexpressed the GOSR1 gene after viral infection.

[0102] (1) Western blot determination of viral protein expression level: After viral infection of cells, cell proteins were extracted and detected by Western blot using viral protein-specific antibodies. The band signals were then quantitatively analyzed to assess the relative expression level of viral proteins. The specific steps are described in Example 1.

[0103] (2) qPCR determination of viral mRNA level: After viral infection of cells, cellular RNA was extracted and RT-qPCR was performed using primers specific to viral M gene mRNA. The relative expression level of viral M gene mRNA was calculated using the relative quantification method. The specific steps are as described in Example 1.

[0104] (3) Immunofluorescence experiment: After the virus infects the cells, the cells are fixed with formaldehyde, and the viral proliferation level is determined using virus protein-specific antibodies and immunofluorescence technology. The specific steps are as described in Example 1.

[0105] Results of tracheal epithelial cells as follows Figure 3A As shown in -D, the WB results show that the tracheal epithelial cells ( Figure 3AOverexpression of the GOSR1 gene increased the relative expression level of NP protein in group H1 from 0.43 to 1.12, while the relative expression level of NP protein in group H5 increased from 0.12 to 0.28. The relative expression level of NP protein in group H1 was significantly higher than that in group H5. p<0.0001). qPCR results showed that after overexpression of GOSR1 in tracheal epithelial cells, the relative expression level of M gene mRNA in group H1 significantly increased from 1 to 3.64 (p<0.0001). p<0.01) Figure 3B The relative expression level of M gene mRNA in group H5 increased significantly from 1 to 3.15. p<0.001) Figure 3C Immunofluorescence results showed that overexpression of GOSR1 significantly enhanced the proliferation level of H1 influenza virus in tracheal epithelial cells. Figure 3D ).

[0106] Results of tracheal fibroblasts as follows Figure 4A As shown in Figure -D, Western blotting results in tracheal fibroblasts showed that overexpression of the GOSR1 gene increased the relative expression level of NP protein in group H1 from 0.05 to 0.45, while the relative expression level of NP protein in group H5 increased from 0.13 to 0.84. The relative expression level of NP protein in group H5 was significantly higher than that in group H1. p<0.001) Figure 4A qPCR results showed that in fibroblasts, overexpression of GOSR1 significantly increased the relative expression level of the M gene mRNA in the H1 group from 1 to 4.63. p<0.01) Figure 4B The relative expression level of M gene mRNA in group H5 increased significantly from 1 to 29.15. p<0.05) Figure 4C Immunofluorescence results showed that overexpression of GOSR1 significantly increased the proliferation levels of H1 and H5 in tracheal fibroblasts. Figure 4D ).

[0107] Example 3

[0108] This example illustrates that a formulation targeting the GOSR1 gene can inhibit the proliferation and spread of influenza A virus in guinea pigs.

[0109] 1. Design small interfering RNA (siRNA) targeting the GOSR1 gene.

[0110] Refer to the design steps in Example 1.

[0111] 2. The GOSR1 gene in guinea pig tracheal epithelial cells was transiently knocked down by siRNA-LNP delivered via intratracheal nebulization.

[0112] Prepare the reagents according to the Invivofectamine™ 3.0 (Invitrogen, IVF3001) kit. Mix 50 µL of 3.6 nmol / µL siRNA with 50 µL of complex buffer, then add 100 µL of Invivofectamine 3.0 reagent. Vortex the resulting mixture vigorously for 2 s, then incubate at 50°C for 30 min. Dilute the mixture with 1 mL of 1×PBS pH 7.4, and finally nebulize 100 μL (i.e., 0.33 μL / g) of the mixture into the trachea of ​​anesthetized 300 g HartLey strain female guinea pigs (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., 4 weeks old) using a nebulizer needle.

[0113] 3. The experiment to detect the aerosol transmissibility of the virus among guinea pigs used HartLey strain female guinea pigs (n=3 per group). Following step 2 above, a mixture of siRNA and LNP was delivered via intratracheal nebulization. The siRNA sequence delivered to the negative control group was the same as the control RNA sequence; the siRNA delivered via nebulization for GOSR1 genome knockdown was as described above. Both the negative control group and the GOSR1 genome knockdown group were inoculated with the aforementioned H1 virus after siRNA delivery via nebulization. The inoculation method was intranasal administration, with a dosage of 10 mg / L for each guinea pig. 6.0 EID 50 After inoculation, the virus-inoculated guinea pigs were placed in cages for 24 hours as donors. 24 hours after inoculation, one donor guinea pig and another new healthy guinea pig (recipient guinea pig, used as the aerosol transmission group) were placed in adjacent cages A and B, respectively. Figure 5 The recipient guinea pigs were housed together for 7 days, spaced 5 cm apart, during which time the recipient guinea pigs did not have direct contact with the infected donor guinea pigs. Nasal wash samples were collected from all guinea pigs on days 1, 3, 5, and 7 after co-hospitalization. The collected samples were diluted 10-fold with PBS buffer containing 1% double antibody, inoculated into SPF-grade chicken embryos, and treated with EID. 50 Viral titration was performed as described in step four below. Serum was collected from all guinea pigs 21 days after inoculation, and hemagglutination inhibition (HI) was performed according to the protocol described in the OIE Terrestrial Animal Diagnostic Tests and Vaccines Manual.

[0114] 4. Experiment to detect the proliferation level of virus in guinea pigs

[0115] (1) Same as the experimental steps in step 3 above, collect three organs (nasal turbinates, trachea and lungs) from guinea pigs on days 1, 3, 5 and 7 after infection, and homogenize each organ for testing;

[0116] (2) Add 800 μL of PBS buffer containing 1% double antibody and 2 sterile steel balls to each of the above guinea pig tissues, place them in a pre-cooled tissue homogenizer (frequency 30 times / s, for a total of 5 min) to homogenize the tissues, then centrifuge at 4°C and 12000 r / min for 5 min, and transfer the supernatant to a new centrifuge tube.

[0117] (3) Dilute each tube of supernatant with PBS buffer containing 1% penicillin and antibiotics for 10 minutes. 1 -10 8 The diluted samples were inoculated into 9-10 day old SPF grade chicken embryos, 200 μL / embryo, 3 embryos for each dilution, and incubated in a 37℃ incubator. Chicken embryos that died nonspecifically after 24 h were discarded, and the remaining chicken embryos were incubated in a constant temperature incubator for 48 h before being collected.

[0118] (4) Determine the hemagglutination status of each chicken embryo and calculate the median infection dose (EID) of the chicken embryo using the internationally accepted Reed-Muench method. 50 .

[0119] The calculation formula is as follows:

[0120] EID 50 =10 X+Y / 200 μL

[0121] X = the logarithm of the viral dilution with a positivity rate just greater than 50%;

[0122] Y = (Percentage of positive rate just above 50% - 50%) / (Percentage of positive rate just above 50% - Percentage of positive rate just below 50%)

[0123] 5. Detection experiment of exhaled viral aerosols in guinea pigs after infection

[0124] The guinea pig infection experiment followed the same steps as in experiment 3 above. On days 1, 3, 5, and 7 post-inoculation, exhaled air from guinea pigs was collected for 60 min at a flow rate of 28.3 L / min using an Anderson six-stage cascade sampler (28.3 L / min × 60 min ≈ 1700 L of sampled air). Viral RNA in the aerosol samples was quantitatively detected by RT-qPCR. The viral concentration in the original samples was calculated based on the standard curve.

[0125] The results are as follows Figure 6A As shown, H1 virus was detected in the nasal wash fluid of all guinea pigs in the negative control group, and the viral titer of H1 in the inoculated group was higher than 10.2.53 EID 50 / mL, the highest viral titer in the aerosol transmission group H1 was 10. 2.20 EID 50 / mL. However, after GOSR1 gene knockdown in the guinea pig trachea, the highest viral titer of H1 in the nasal wash fluid of the vaccinated group was 10 on day 3, at which point the titer was 10. 3.03 EID 50 / mL, the lowest viral titer of H1 was 10 on day 7. 1.20 EID 50 / mL; H1 virus was not detected in nasal wash fluid from all guinea pigs in the aerosol transmission group ( Figure 6B The results of the antibody assay on guinea pig serum are as follows: Figure 6C The results showed that antibodies were detectable in the serum of all guinea pigs in the negative control group, with the lowest antibody titer in the inoculated group being 2. 7 The antibody titer in the aerosol transmission group was 2. 6 -2 8 After knocking down the GOSR1 gene in the guinea pig trachea, antibodies were detectable in the serum of the inoculated group of guinea pigs, with an antibody titer of 2. 4 -2 5 Antibodies were not detected in the serum of guinea pigs in all aerosol transmission groups. These results indicate that H1 can be transmitted among guinea pigs via aerosols. However, after the GOSR1 gene was knocked down in the guinea pig trachea, H1 could not be transmitted among guinea pigs via aerosols, thus reducing the transmission efficiency of H1 from 100% to 0%.

[0126] Guinea pig tissue virus titer test results as follows Figure 6D and 6E The results showed that the highest viral titers of H1 in the nasal turbinates, trachea, and lung tissues of guinea pigs in the negative control group were 10, respectively. 5.78 10 3.20 and 10 5.20 EID 50 / mL ( Figure 6D ); and after targeted knockdown of the GOSR1 gene in the guinea pig trachea, the highest viral titers of H1 in the guinea pig nasal turbinates, trachea, and lung tissue decreased to 10, respectively. 2.2 10 1.2 and 10 2.45 EID 50 / mL ( Figure 6E The above results indicate that high viral titers can be detected in the nasal turbinates, trachea, and lung tissues of guinea pigs infected with H1. After the GOSR1 gene was knocked down in the guinea pig trachea, the viral titer levels in all three tissues decreased. Knocking down the GOSR1 gene can reduce the viral proliferation level in different tissues of guinea pigs.

[0127] Results of exhaled viral aerosol experiments as follows Figure 6F As shown, the highest viral concentration in the exhaled aerosols of guinea pigs infected with H1 was 10 on day 3. 7.38 Copy count / L, minimum 10 on day 7 5.55 Copy number / L; and after GOSR1 gene knockdown in guinea pig trachea, the highest viral concentration in exhaled aerosols of guinea pigs was 10 on day 1. 3.36 Copy number / L, no H1 virus detected on day 7. These results show that targeted knockdown of the GOSR1 gene in the guinea pig trachea can significantly reduce the viral concentration in the exhaled aerosol of guinea pigs.

[0128] In conclusion, knocking down the GOSR1 gene in the trachea of ​​guinea pigs can significantly inhibit the proliferation and spread of influenza A virus in guinea pigs.

[0129] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. The use of a preparation containing a knockdown of the GOSR1 gene expression in tracheal epithelial cells and / or tracheal fibroblasts in the preparation of a drug for the prevention and / or treatment of influenza A virus H1 and / or influenza A virus H5 infection, wherein the preparation is a nucleic acid drug selected from siRNA, the sense strand sequence of the siRNA being shown in SEQ ID NO: 1 and the antisense strand sequence being shown in SEQ ID NO:

2.

2. The application according to claim 1, wherein the formulation is used to inhibit the proliferation of influenza virus.

3. The application according to claim 1, wherein the formulation is used to reduce influenza virus protein expression.

4. The application according to claim 3, wherein, The protein includes at least one of nucleoprotein, hemagglutinin, neuraminidase, RNA polymerase, basic protein, matrix protein, and non-structural high protein.

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