Use of sectm1 protein in preparation of medicine for treating influenza

Abstract

This invention relates to the field of gene therapy, specifically to the application of SECTM1 protein in the preparation of drugs for treating influenza. This invention reveals for the first time that SECTM1 has a significant inhibitory effect on influenza virus infection and replication, expanding the application scope of interferon-stimulated genes in the antiviral field; it demonstrates that SECTM1 has inhibitory effects on different subtypes of influenza viruses, possessing potential broad-spectrum antiviral application value; it demonstrates that SECTM1 can exert its antiviral effect through secretory supernatant or exogenous addition of recombinant protein, breaking through the limitation of traditional host-restricting factors acting only within cells; and it verifies the in vivo antiviral effect of SECTM1 in animal models, providing reliable experimental evidence for its clinical translation and application.

Description

Technical Field

[0001] This invention relates to the field of gene therapy, specifically to the application of SECTM1 protein in the preparation of drugs for treating influenza. Background Technology

[0002] Influenza viruses are a group of respiratory pathogens that seriously endanger human health, especially influenza A virus (IAV). Their genomes are prone to mutation and reassortment, leading to the continuous emergence of different subtypes, resulting in high transmissibility and a potential pandemic risk. Existing antiviral drugs mainly include neuraminidase inhibitors, M2 ion channel inhibitors, and polymerase inhibitors developed in recent years. However, these drugs generally suffer from problems such as rapid development of drug resistance, single target, or limited therapeutic window, severely restricting their clinical application effectiveness.

[0003] Host-derived antiviral factors, especially interferon-stimulated genes (ISGs), play a crucial role in innate immune defense. Some ISGs can inhibit viral infection by interfering with multiple stages of viral entry, replication, assembly, or release, and are therefore considered important candidate targets for developing novel antiviral strategies. However, the number of host factors with clearly defined broad-spectrum anti-influenza virus activity remains limited, and their mechanisms of action and application are still unclear.

[0004] SECTM1 (Secreted and Transmembrane Protein 1) is a type I transmembrane secretory protein, also known as K12, characterized by type 1a transmembrane proteins of the SECTM family. It exhibits a perinuclear Golgi apparatus-like distribution and is believed to be involved in hematopoietic and / or immune system processes. Human K12 protein has been shown to be primarily expressed in the spleen, prostate, testes, small intestine, and peripheral blood leukocytes. K12 protein expression on the cell surface is extremely low and undetectable. Alternatively, K12 on the cell surface is rapidly cleaved, generating soluble K12 protein. Immunohistochemical analysis of peripheral blood cells shows that K12 is present in myeloid leukocytes, with the most prominent staining in granulocytes, but no detectable expression in lymphocytes. It may be involved in thymocyte signaling. Some researchers have proposed a role for K12 produced in the thymic microenvironment in regulating thymocyte signaling and cytokine release, particularly in pathological environments of the thymus, such as IFN-γ upregulation in myasthenia gravis. Furthermore, as a presumed natural CD7 ligand, SECTM1 / K12 may be responsible for its co-stimulatory role in T cell activation. However, the role of SECTM1 in viral infection, especially its function and application value in influenza virus infection, remains unclear. Current technology has not revealed whether SECTM1 and its soluble form have a direct inhibitory effect on influenza virus infection or replication, nor have any related technical solutions been found for its in vitro and in vivo application as an anti-influenza virus factor. Therefore, it is urgent to elucidate its mechanism of action in order to provide new technical means for the prevention and control of influenza virus infection. Summary of the Invention

[0005] Addressing a technological gap in existing technologies, this invention discloses for the first time that SECTM1 has a significant inhibitory effect on influenza virus infection and replication, expanding the application scope of interferon-stimulated genes in the antiviral field. The specific details of this invention are as follows: In a first aspect, the present invention provides the application of the SECTM1 protein in the preparation of antiviral drugs.

[0006] Furthermore, the antiviral drug is an antiviral drug for influenza viruses. Optionally, the influenza virus includes influenza A virus and / or influenza B virus.

[0007] Furthermore, the influenza virus is influenza A. Preferably, the influenza A virus includes influenza viruses with different hemagglutinin and neuraminidase subtypes.

[0008] Furthermore, the application includes any of the following aspects: The application of A1.SECTM1 gene as a target in screening drugs for the prevention, mitigation and / or treatment of influenza; The use of A2.SECTM1 protein in the preparation of drugs for the prevention, relief and / or treatment of influenza.

[0009] Furthermore, the drug described in A1 targets the SECTM1 gene and upregulates the expression of the SECTM1 gene.

[0010] The SECTM1 protein exerts its anti-influenza virus effect through any of the following mechanisms: B1. A soluble protein expressed by cells and secreted into the culture supernatant; B2. Recombinant protein added exogenously.

[0011] Furthermore, the SECTM1 gene exerts its antiviral effect by inhibiting at least one step in the viral infection process, namely, viral entry into cells, viral replication, or viral transmission.

[0012] In a second aspect, the present invention provides the use of the SECTM1 gene in the preparation of gene therapy drugs for the treatment or prevention of influenza virus infection.

[0013] Furthermore, the gene therapy drug comprises an expression vector encoding a nucleic acid sequence of the SECTM1 protein.

[0014] Furthermore, the expression vector is an adenovirus vector or a lentivirus vector.

[0015] In a third aspect, the present invention provides a medicament for treating or preventing influenza virus infection, characterized in that the medicament comprises: C1. An effective amount of SECTM1 protein, and a pharmaceutically acceptable carrier or excipient; or C2. An effective amount of an expression vector containing a nucleic acid sequence encoding the SECTM1 protein, and a pharmaceutically acceptable vector or excipient.

[0016] Furthermore, the SECTM1 protein is a recombinant SECTM1 protein.

[0017] Furthermore, the expression vector is an adenovirus vector or a lentivirus vector.

[0018] In one specific embodiment of the present invention, the SECTM1 gene is introduced into the lungs via an adenovirus vector or a lentivirus vector for stable expression, thereby achieving the purpose of treating influenza.

[0019] In a fourth aspect, the present invention provides a system for screening drugs for the prevention, mitigation, and / or treatment of influenza, the system screening drugs for the prevention, mitigation, and / or treatment of influenza by determining whether the drug can promote the expression of the SECTM1 gene.

[0020] The beneficial effects of the present invention include, but are not limited to: This invention reveals for the first time that SECTM1 has a significant inhibitory effect on influenza virus infection and replication, expanding the application scope of interferon-stimulated genes in the field of antiviral therapy. This invention demonstrates that SECTM1 has an inhibitory effect on different subtypes of influenza virus and has potential broad-spectrum anti-influenza virus application value; This demonstrates that SECTM1 can exert its antiviral effect through secretory supernatant or exogenous addition of recombinant protein, breaking through the limitation of traditional host restriction factors that are limited to intracellular action. This invention validated the in vivo anti-influenza virus effect of SECTM1 in animal models, providing reliable experimental evidence for its clinical translation and application. Attached Figure Description

[0021] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is a schematic diagram of the results of Western Blot analysis of the inhibition of influenza virus protein expression levels by overexpressing the SECTM1 gene in an embodiment of the present invention, wherein the influenza A virus NP (nuclear protein), influenza A virus NS1 (non-structural protein), influenza A virus M1 (matrix protein), influenza A virus surface glycoprotein HA (hemagglutinin), and influenza A A / WSN / 33 (H1N1) are represented.

[0022] Figure 2 This is a schematic diagram illustrating the results of plaque assay for determining viral titer in an embodiment of the present invention, showing the viral replication level of the overexpressing SECTM1 gene. Figure 2 A in the diagram shows the plaque staining results that inhibit influenza virus replication. Figure 2 Figure B shows the viral titer statistics.

[0023] Figure 3 This is a schematic diagram of SECTM1 knockout protein expression in A549 cells in an embodiment of the present invention.

[0024] Figure 4 This is a schematic diagram illustrating how the deletion of the SECTM1 gene promotes protein expression levels in different subtypes of influenza virus in an embodiment of the present invention.

[0025] Figure 5 This is a schematic diagram showing the results of SECTM1 secretory supernatant inhibiting the protein expression level of influenza virus in an embodiment of the present invention.

[0026] Figure 6This is a schematic diagram of the results of plaque assay for determining the viral titer in an embodiment of the present invention, showing the level of influenza virus replication inhibition by SECTM1 secretion supernatant.

[0027] Figure 7 This is a schematic diagram showing the results of the recombinant protein SECTM1 inhibiting the protein expression level of influenza virus in an embodiment of the present invention.

[0028] Figure 8 This is a schematic diagram illustrating the viral delivery protocol of SECTM1 to mice and the influenza virus infection protocol in this embodiment of the invention. It includes intratracheal delivery (0-18 days), intranasal infection with PR8 virus (75 PFU), and subsequent 14-day monitoring of body weight and survival (n = 14 mice / group). On day 6 after PR8 virus infection, lung tissue was taken for viral nucleic acid load detection.

[0029] Figure 9 This diagram illustrates the results of SECTM1 overexpression alleviating weight loss in mice caused by influenza A virus infection in an embodiment of the present invention. Daily weight changes in mice of each group were recorded over 14 days post-infection and expressed as normalized initial weight (Mean ± SEM); the x-axis represents the number of days since influenza PR8 infection, and the y-axis represents the percentage of mouse body weight.

[0030] Figure 10 This diagram illustrates the results of SECTM1 overexpression completely protecting mice from death caused by influenza infection in an embodiment of the present invention. Kaplan-Meier survival curves were used. The cumulative survival of mice in each group over 14 days was recorded, and statistical analysis was performed using the Log-rank (Mantel-Cox) test. The horizontal axis represents the number of days since influenza virus PR8 infection, and the vertical axis represents the survival rate of the mice.

[0031] Figure 11 This diagram illustrates the results of SECTM1 overexpression reducing viral load in mouse lung tissue in this embodiment of the invention. Analysis of viral nucleic acid (vRNA) load in lung tissue: Total RNA was extracted from the lung tissue of mice in each group on day 6 post-infection. Viral RNA levels were detected by RT-qPCR and normalized to the control group (Mean ± SD). Detailed Implementation

[0032] The present invention is described in detail below with reference to the embodiments, but the present invention is not limited to these embodiments. Unless otherwise specified, the raw materials and catalysts in the embodiments of the present invention are all purchased through commercial channels.

[0033] The IAVs involved in this invention cover representative strains of multiple subtypes, including: human seasonally prevalent strains A / WSN / 33 (H1N1), A / Brisbane / 10 / 2007 (H3N2), and Influenza A / Puerto Rico / 8 / 1934 (PR8), all of which originated from the Institute of Pathogenic Biology, Chinese Academy of Medical Sciences; avian low pathogenicity strain A / Quail / Hong Kong / G1 / 1997 (H9N2), which originated from the Institute of Microbiology, Chinese Academy of Sciences; and A / Vietnam / 1203 / 2004 (H5N1, HALo), a genetically modified low pathogenicity virus strain, which originated from the Mount Sinai School of Medicine in the United States.

[0034] All related experiments were conducted in strict accordance with the "Regulations on Biosafety Management of Pathogenic Microorganism Laboratories" and related standards. In vitro infection experiments were all completed under biosafety level 2 (BSL-2) laboratory conditions.

[0035] Example 1: In vitro validation of the inhibitory effect of SECTM1 on influenza virus replication (I) Construction of SECTM1-3×FLAG recombinant expression plasmid This embodiment uses the human SECTM1 gene as the research object, constructs its eukaryotic expression plasmid and lentiviral expression plasmid for subsequent cell-level verification of anti-influenza virus function.

[0036] Specifically, the nucleotide sequence encoding the full-length SECTM1 protein was obtained (Gene ID: 6398, Swiss Prot: ). Q8WVN6 Primers were designed based on the multiple cloning sites of the target vectors (eukaryotic expression vector pMO2 and lentiviral expression vector FG-EH-DEST2-PGK-Puro-WPRE, preserved in our laboratory). To achieve C-terminal FLAG tag fusion, a linker peptide (SGSG) and a 3×FLAG coding sequence were introduced into the downstream primers. EcoRI and Xho I restriction enzyme sites were introduced flanking the eukaryotic expression plasmid primers, respectively; Asc I and Mlu I restriction enzyme sites were introduced flanking the lentiviral plasmid primers. A FLAG-tagged expression plasmid was constructed using a combination of PCR amplification and homologous recombination.

[0037] amino acid sequence: MQTCPLAFPGHVSQALGTLLFLAASLSAQNEGWDSPICTEGVVSVSWGENTVMSCNISNAFSHVNIKLRAHGQESAIFNEVAPGYFSRDGWQLQVQGGVAQLVIKGARDSHAGLYMWHLVGHQRNNRQVTLEVSGAEPQSAPDTGFWPVPAVVTAVFILLVALVMFAWYRCRCSQQRREKKFFLLEPQMKVAALRAGAQQGLSRASAELWTPDSEPTPRPLALVFKPSPLGALELLSPQPLFPYAADP (SEQ ID NO. 1).

[0038] DNA sequence: (SEQ ID NO.2).

[0039] The specific steps are as follows: Using a template plasmid containing SECTM1 as the amplification template, specific primers targeting the upstream and downstream of the SECTM1 coding region were designed to amplify the target gene SECTM1 by PCR. In the initial amplification stage, only the aforementioned upstream and downstream primers were added, and 15 amplification cycles were performed to obtain a specific amplified fragment of the target gene SECTM1.

[0040] Subsequently, primers containing the FLAG tag sequence and the homologous arm sequence of the target gene SECTM1 were added to the PCR reaction system. Amplification was performed for 30 more reactions based on the original amplification system, so that the FLAG tag sequence fused with the coding sequence of the target gene SECTM1 through the homologous arm, thereby obtaining an amplification product with the FLAG tag fused at the C-terminus.

[0041] The primers were constructed as shown in Table 1.

[0042] Table 1 Primer sequences for the SECTM1 gene

[0043] After the amplification reaction, the products were identified by 1% agarose gel electrophoresis and then recovered using the TIANGEN universal DNA purification kit. The recovered SECTM1-3×FLAG (abbreviated as SECTM1-3F) fragment was double-digested with the corresponding vector backbone. EcoRI and XhoI were used in the eukaryotic expression system, and AscI and MluI were used in the lentiviral system. After recovering the corresponding DNA fragments by agarose gel electrophoresis, the SECTM1 fragment was ligated into the double-digested linearized vector using DNA ligase, thereby constructing the SECTM1-3F recombinant expression plasmid.

[0044] The amino acid sequence of SECTM1-3F: the linker is KLSGSG (underlined part), and the 3× tag sequence is DYKDHDGDYKDHDIDYKDDDDK (bold part).

[0045] MQTCPLAFPGHVSQALGTLLFLAASLSAQNEGWDSPICTEGVVSVSWGENTVMSCNISNAFSHVNIKLRAHGQESAIFNEVAPGYFSRDGWQLQVQGGVAQLVIKGARDSHAGLYMWHLVGHQR NNRQVTLEVSGAEPQSAPDTGFWPVPAVVTAVFILLVALVMFAWYRCRCSQQRREKKFFLLEPQMKVAALRAGAQQGLSRASAELWTPDSEPTPRPLALVFKPSPLGALELLSPQPLFPYAADP KLSGSG DYKDHDGDYKDHDIDYKDDDDK (SEQ ID NO. 3).

[0046] DNA sequence of SECTM1-3F: the linker is AAGCTTAGCGGCAGTGG (underlined portion), and the 3×flag tag sequence is AGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAGTAG (boldfaced portion).

[0047] ATGCAGACCTGCCCCCTGGCATTCCCTGGCCACGTTTCCCAGGCCCTTGGGACCCTCCTGTTTTTGGCTGCCTCCTTGAGTGCTCAGAATGAAGGCTGGGACAGCCCCATCTGCACAGAGGGGGTAGTCTCTGTGTCTTGGGGCGAGAACACCGTCATGTCCTGCAACATCTCCAACGCCTTCTCCCATGTCAACATCAAGCTGCGTGCCCACGGGCAGGAGAGCGCCATCTTCAATGAGGTGGCTCCAGGCTACTTCTCCCGGGACGGCTGGCAGCTCCAGGTTCAGGGAGGCGTGGCACAGCTGGTGATCAAAGGCGCCCGGGACTCCCATGCTGGGCTGTACATGTGGCACCTCGTGGGACACCAGAGAAATAACAGACAAGTCACGCTGGAGGTTTCAGGTGCAGAACCCCAGTCCGCCCCCGACACTGGGTTCTGGCCTGTGCCAGCGGTGGTCACTGCTGTCTTCATCCTCTTGGTCGCTCTGGTCATGTTCGCCTGGTACAGGTGCCGCTGTTCCCAGCAACGCCGGGAGAAGAAGTTCTTCCTCCTAGAACCCCAGATGAAGGTCGCAGCCCTCAGAGCGGGAGCCCAGCAGGGCCTGAGCAGAGCCTCCGCTGAACTGTGGACCCCAGACTCCGAGCCCACCCCAAGGCCGCTGGCACTGGTGTTCAAACCCTCACCACTTGGAGCCCTGGAGCTGCTGTCCCCCCAACCCTTGTTTCCATATGCCGCAGACCCA AAGCTTAG CGGCAGTGGAGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAGTAG (SEQ ID NO. 4).

[0048] The ligation product was transformed into competent *E. coli*, and positive clones were screened. Restriction endonuclease digestion and nucleotide sequencing confirmed the correct insertion sequence and complete reading frame. The correctly identified recombinant plasmid was used for subsequent cell transfection and anti-influenza virus experiments.

[0049] (II) Construction of SECTM1 knockout plasmid (sgRNA) Using CRISPR / Cas9 technology, a SECTM1 gene knockout plasmid was constructed by cloning a specific sgRNA into the lentivirus expression vector LentiCRISPRv2 containing mCherry fluorescent labeling.

[0050] (1) sgRNA design and primer annealing Specific targeting sgRNAs were designed based on the SECTM1 gene sequence. Primers were synthesized by Anshengda Biotechnology Co., Ltd., and the primer sequences are shown in Table 2.

[0051] Table 2 SECTM1 sgRNA Primer Sequences

[0052] The synthesized upstream and downstream primers (10 μM) were mixed in equal volumes and annealed using a gradient cooling method to form a double-stranded sgRNA insert fragment.

[0053] Annealing procedure: First, heat at 70 °C for 20 min to fully denature the primers, then cool to 37 °C and maintain for 30 min to promote the formation of a stable double-stranded structure by complementary base pairing of the upstream and downstream primers. The product is then placed on ice for later use.

[0054] (2) Linearization of lentiviral CRISPR vectors The LentiCRISPRv2-mCherry vector was linearized using the restriction endonuclease BsmB I (see Table 3 for the digestion system), resulting in sticky ends complementary to the sgRNA fragment. The digestion reaction system is also shown in Table 3. The reaction was incubated at 55 °C for 1 h, followed by separation of the products by 1% agarose gel electrophoresis. The linearized vector fragment was then recovered using a DNA purification kit.

[0055] Table 3 LentiCRISPRv2-mCherry vector digestion system

[0056] (3) Plasmid ligation and clone identification: The linearized vector backbone was ligated to the annealed double-stranded sgRNA fragment using T4 DNA Ligase. The ligation system is shown in Table 4. The reaction was carried out at room temperature (25 °C) for 2 h.

[0057] Table 4. sgRNA-vector ligation reaction system

[0058] The ligation product was transformed into DH5α competent E. coli cells and plated on LB agar plates containing ampicillin for overnight culture. The following day, single clones were randomly selected for amplification, plasmids were extracted, and sent to the company for Sanger sequencing. After sequence alignment confirmed correct sgRNA insertion and the absence of mutations, glycerol-containing bacteria were prepared and stored at -80 °C.

[0059] (III) Methods for constructing A549 cell lines with stable overexpression or knockout of SECTM1 In this embodiment, a stable human lung epithelial cell line A549 with SECTM1 overexpression or knockout was constructed using lentivirus-mediated methods.

[0060] The constructed lentiviral expression plasmid FG-EH-DEST2-SECTM1-3F or the CRISPR / Cas9 system lentiviral knockout plasmid LentiCRISPRv2-mCherry-SECTM1 sgRNA were mixed with packaging helper plasmids psPAX2 and pMD2.G at a mass ratio of 4:3:2, and then co-transfected into 293t cells using transfection reagents for lentiviral packaging. The cell culture supernatant was collected 48 hours after transfection. This supernatant contained recombinant lentiviral particles and was used directly after removing cell debris by low-speed centrifugation (1000 rpm, 3 minutes), or frozen at -80°C for subsequent cell infection experiments.

[0061] The obtained lentiviral supernatant was added to A549 cells for infection. Forty-eight hours after infection, puromycin was added to the culture medium for resistance selection to identify cells successfully integrated with the lentiviral vector. Two to three rounds of resistance selection and cell expansion were performed to obtain two cell lines: a stable SECTM1 overexpressing A549 cell line (A549-SECTM1OE) and a SECTM1 gene knockout A549 cell line (A549-SECTM1 KO). Stable expression of the SECTM1 protein was confirmed by Western blot.

[0062] Meanwhile, a stable A549 cell line infected with an empty vector lentivirus was constructed using the same method as a control group.

[0063] (iv) Influenza A / WSN / 33 (H1N1) infection A549-WT cells and A549-SECTM1 OE cells were seeded in 12-well plates, with 2.5 × 10⁶ cells per well. 5 After culturing the cells overnight, the cell culture supernatant was discarded, and the cells were gently washed once with sterile PBS solution. Then, influenza virus inoculation solution was added to the cells, and infection was carried out at a multiplicity of infection (MOI) of 0.01. The cells were incubated at 37 °C for 1 hour to ensure that the virus fully adsorbed onto the cells.

[0064] After the virus adsorption was complete, the virus inoculation solution was discarded, and the cells were washed again with PBS solution. Then, growth maintenance medium containing trypsin was added, and the cells were cultured at 37 °C and 5% CO2.

[0065] (v) Detection of viral protein expression levels Cells were collected and lysed with cell lysis buffer after 24, 36, 48, and 72 hours of viral infection, respectively, to obtain total cellular protein samples. The expression levels of influenza virus-related proteins were detected by SDS-PAGE electrophoresis and Western blot to assess the inhibitory effect of SECTM1 on influenza virus protein expression. The antibodies used for WB detection are shown in Table 5.

[0066] Table 5 Antibodies used in WB detection

[0067] Simultaneously, the cell culture supernatant after infection was collected, aliquoted, and then... Store at 80 °C. The supernatant sample was used for subsequent viral replication capacity testing, including viral titer determination by plaque assay, to further verify the inhibitory effect of SECTM1 on influenza virus replication.

[0068] (vi) Plaque assay to determine viral titer at viral replication level Experimental principle of viral plaque assay Virus solutions of various dilutions were inoculated into a monolayer cell culture environment. After adsorption for 2 hours, agarose was applied to the monolayer cells. The virus infected and multiplied within the cells, causing them to rupture and die. Due to the limitations of the solid medium, the released virus could only spread outwards from the initially infected cells. After several proliferation cycles, a localized lesion area, known as a viral plaque, was formed. After crystal violet staining, normal cells appeared purple, while the plaque area remained unstained, forming an unstained region.

[0069] Experimental materials: MDCK cells and cell culture reagents; 4% tissue fixative (Solepro, #P1110-500ml); Crystal violet.

[0070] Viral infection fluid: DMEM 48.5 mL; 7.5% BSA 500 μL; PS500 μL; TPCK-Trypsin (1 mg / mL) 50 μL.

[0071] 2% Low Melting Point Agarose: 2g of low-melting-point agarose; 100mL of deionized water; Store at room temperature after autoclaving. (Note: Alternatively, you can prepare the solution using autoclaved deionized water first, then boil it without further autoclaving.) Experimental steps: The day before, MDCK cells were digested with EDTA-Trypsin, counted, and seeded in 12-well plates at a cell concentration of 2 × 10⁶ cells / well. 5 / well, incubate at 37℃ in a 5% CO2 incubator for 24 hours.

[0072] Aspirate the cell culture supernatant from the plate and wash the plate twice with 500 µL PBS per well.

[0073] Add 300 µL of diluted Influenza A / WSN / 33 (H1N1) virus solution to each well, with two parallel wells for each dilution. (Sample dilution: Add 40 µL of sample to 360 µL of virus infection solution for a 10-fold dilution).

[0074] Place the cell plate in a 37 ℃ 5% CO2 incubator to allow the virus to adsorb for 1-2 h.

[0075] After adsorption for 1-2 hours, remove the supernatant. Wash the plate once with 500 µL / well of PBS.

[0076] After microwaving 2% low-melting-point agarose, place it in a 50°C water bath and incubate at 37°C with 2×DMEM (2% PS, 1% FBS, 2µg / mL TPCK-Trypsin). Mix 2% low-melting-point agarose and 2×DMEM (2% PS, 1% FBS, 2µg / mL TPCK-Trypsin) in a 1:1 ratio, and then add 1.5 mL of the mixture to each well of a 12-well plate.

[0077] Place the cell culture plate in a biosafety cabinet until the gel solidifies, then invert it and incubate it in a 37°C 5% CO2 incubator for 3 days.

[0078] Once white spots appear, add 1 mL of 4% tissue fixative per well to a 12-well plate and incubate overnight at 4°C.

[0079] After fixing the plate overnight, rinse off the gel under running water and add 1 mL of crystal violet per well for staining.

[0080] Remove the staining solution, rinse with running water, dry, and then photograph the counting plate.

[0081] Result determination: The calculation of the vacuole formation unit is as follows:

[0082] The calculated result is the reference titer for this batch of virus.

[0083] If the dilution is 10 -3 The average number of empty plates is 40, and the infection volume is 300 μL. Therefore, the result is (40 × 10⁻⁶). 3 pfu) / 0.3 mL = 1.3 × 10 5 pfu / mL.

[0084] from Figure 1 , Figure 2 It can be seen that overexpression of SECTM1 significantly reduced the expression level of viral proteins and the viral replication level.

[0085] Specific Example 2: Inhibitory Effect of SECTM1 on Different Subtypes of Influenza Viruses In this embodiment, A549-WT and A549-SECTM1 KO cells were infected with different subtypes of influenza virus: A / WSN / 33 (H1N1), A / Brisbane / 10 / 2007 (H3N2), A / Quail / Hong Kong / G1 / 1997 (H9N2) and A / Vietnam / 1203 / 2004 (H5N1), all of which were preserved in our laboratory.

[0086] A549-WT and A549-KO cells were seeded in 12-well plates. After the cells reached 60% confluence, they were infected with H1N1, H3N2, H5N1, and H9N2 viruses with an MOI of 0.1, respectively. Simultaneous infection was performed using the same strategy of adsorption at 4 °C for 1 h. After changing the maintenance medium, the cells were cultured for 24 h.

[0087] Cell lysates from each group were collected, and the expression abundance differences of different subtype viral proteins in the SECTM1-deficient background were detected by Western blot to assess the sensitivity and regulatory role of SECTM1 in different subtypes of IAV.

[0088] from Figure 3 The results showed that SECT1 protein expression was not detected in A549-SECTM1 KO cells, indicating that SECT1 had been completely knocked out. Figure 4 Western blot (WB) results showed that, compared with WT control cells, the expression levels of NP, NS1, and M1 proteins of different influenza virus subtypes were significantly increased in SECTM1-deficient cells. This phenomenon was consistent across all subtypes, suggesting that SECTM1 has an inhibitory effect on influenza A viruses of different origins and antigenic subtypes.

[0089] The results in summary indicate that the inhibitory effect of SECTM1 on influenza A virus replication is not limited to specific subtypes, but rather exhibits certain broad-spectrum anti-influenza virus characteristics.

[0090] Specific Example 3: In vitro validation of the inhibitory effect of SECTM1 secretory supernatant on influenza virus (a) Preparation of SECTM1 secretory supernatant In this embodiment, human embryonic kidney cells 293T were used as the cells for preparing SECTM1 secretory supernatant. 293T cells were cultured in DMEM medium containing 10% fetal bovine serum at 37 ℃ and 5% CO2. When the cells reached 70–80% confluence, they were transfected with the SECTM1 expression plasmid, the empty vector control plasmid, and the positive control CH25H (Gene ID: 9023, Swiss Prot: O95992) expression plasmid, respectively.

[0091] Transfection was performed using PEI-Max transfection reagent, with 10 culture dishes transfected for each plasmid to ensure sufficient supernatant. Six to eight hours after transfection, the original culture medium was discarded and replaced with Opti-MEM serum-free medium for further culture.

[0092] Forty-eight hours post-transfection, cell culture supernatant was collected and centrifuged at low speed (approximately 1000 rpm) to remove cell debris, yielding a clear supernatant. The supernatant was then concentrated using an ultrafiltration tube with a molecular weight cutoff of 15 kDa, reducing approximately 100 mL to a suitable volume. The concentrated supernatant was then aliquoted and stored in [specific container / system]. Store at 80℃ for later use.

[0093] (ii) Incubation treatment of secretory supernatant with influenza virus In the antiviral verification experiment, the SECTM1 secretion supernatant, the empty vector control supernatant, and the positive control CH25H supernatant were mixed with an equal amount of influenza virus and incubated at 37 °C for about 1 hour to allow the secretion products to come into full contact with the virus.

[0094] (III) Detection of the inhibitory effect of secretory supernatant on influenza virus infection and replication The virus treated as described above was used to infect susceptible host cells A549. After the virus adsorption process was completed at 37 °C, the culture medium was replaced with growth maintenance medium for further culture.

[0095] Twenty-four hours after infection and culture, cell samples were collected, and the expression levels of influenza virus-related proteins were detected by Western blot to assess the inhibitory effect of SECTM1 secretion supernatant on viral protein expression. Simultaneously, post-infection cell culture supernatant was collected, aliquoted, and... Store at 80 °C. The supernatant sample was used for subsequent viral replication capacity testing, including viral titer determination by plaque assay, to further verify the inhibitory effect of SECTM1 on influenza virus replication.

[0096] from Figure 5 , Figure 6 It can be seen that incubating the cell culture supernatant expressing SECTM1 with influenza virus can significantly reduce the virus's ability to infect host cells.

[0097] Specific Example 4: Verification of the Inhibitory Effect of SECTM1 Recombinant Protein on Influenza Virus in Vitro (a) Sources and processing of SECTM1 recombinant protein The recombinant SECTM1 protein used in this embodiment is commercially available recombinant human SECTM1 protein, purchased from Ibotek, catalog number RP00278. This protein was purified by the manufacturer and dissolved in a suitable buffer solution, then aliquoted and stored according to the instructions. Thaw slowly on ice before use at 80 ℃.

[0098] Before use in the experiment, the SECTM1 recombinant protein was serially diluted with sterile phosphate-buffered saline (PBS) or serum-free culture medium for cell culture to prepare working solutions of different concentrations for subsequent in vitro antiviral experiments.

[0099] (ii) Incubation treatment of SECTM1 recombinant protein with influenza virus Influenza A / WSN / 33 (H1N1) virus with an MOI of 0.1 was mixed with working solutions of recombinant protein of different concentrations of SECTM1 (0, 0.5 ug / mL, 1 ug / mL) and incubated at 37 °C for 30-60 minutes to allow the recombinant protein to come into full contact with the virus.

[0100] (III) Detection of the inhibitory effect of SECTM1 recombinant protein on influenza virus infection The virus, after the above treatment, was used to infect susceptible host cells such as A549 cells. One hour after virus adsorption, the virus inoculum was discarded, and the cells were cultured again using growth maintenance medium.

[0101] Cell samples were collected 24 hours after infection and culture, and the expression levels of influenza virus-related proteins were detected by Western blot to assess the inhibitory effect of SECTM1 recombinant protein on viral protein expression.

[0102] from Figure 7 It can be seen that the exogenous addition of SECTM1 recombinant protein also has a significant inhibitory effect on influenza virus infection or replication.

[0103] The above results indicate that SECTM1 can not only act as an intracellular antiviral factor, but also exert antiviral effects through secretion or exogenous administration.

[0104] Specific Example 5: Inhibitory Effect of Recombinant Adeno-Associated Virus-Mediated SECTM1 on Influenza Virus Infection in Mice The experimental flowchart of the inhibitory effect of recombinant adeno-associated virus-mediated SECTM1 on influenza virus infection in mice is shown below. Figure 8 As shown.

[0105] (a) Laboratory animals and grouping Healthy SPF-grade mice were selected as experimental animals and acclimatized under standard feeding conditions before the experiment. According to the experimental design, the mice were randomly divided into the following groups: 1) Control group (AAV-Vector): Recombinant virus carrying empty vector was injected via trachea.

[0106] 2) Experimental group (AAV-SECTM1): Recombinant virus carrying human SECTM1 gene was injected via tracheal intubation.

[0107] Each group consisted of 28 mice, with all groups maintaining consistency in sex, weight, and other characteristics.

[0108] (ii) In vivo expression of SECTM1 delivered by adenovirus Recombinant adeno-associated virus (AAV) was used as the delivery vector (AAV-CASI Promoter-ZsGreen-WPRE). The human SECTM1 coding sequence (CDS) was cloned into a CASI-driven expression framework with a strong promoter, and a 3×Flag tag was fused to its C-terminus for subsequent immunological detection. ZsGreen fluorescent protein was co-expressed as a reporter gene via a p2A self-cleaving peptide (AAV-CASI Promoter-SECTM1-3x flag-p2A-ZsGreen-WPRE). The plasmid construction, virus packaging, purification, and titer determination of the above AAV vector (serotype: AAV6.2ff) were commissioned to Heyuan Biotechnology (Shanghai) Co., Ltd.

[0109] Specific procedures for inoculating mice with recombinant adeno-associated virus: Mice were deeply anesthetized (without corneal reflex and no response to pain tests) by intraperitoneal injection of 300 μL of ready-to-use tribromoethanol anesthetic. They were then fixed in a supine position on a surgical board, and their necks were prepared and disinfected with 75% alcohol. The skin of the mouse's neck was cut approximately 1 cm open with sterile scissors, and the recombinant adeno-associated virus (20 μL x 10⁻¹) was administered via tracheal injection. 11 The adenovirus (vg / mouse) was delivered to mice to induce SECTM1 expression in respiratory tissues; the wounds were sutured, and the mice were observed the following day. Control mice received the same dose of empty vector adenovirus via the same method. After adenovirus delivery, the mice were fed for 18 days to ensure adequate expression of exogenous SECTM1 in vivo.

[0110] (iii) Influenza virus infection On day 18 after adenovirus delivery, influenza virus A / Puerto Rico / 8 / 1934(H1N1) (PR8) was administered intranasally to mice in each group to establish an influenza virus infection model. The viral dose was 75 pfu per mouse. Mice were randomly assigned to the following groups according to the experimental design: 1) AAV-Vector + PBS group: Empty vector virus was pre-injected, and an equal volume of sterile PBS was dripped during the challenge experiment as a blank control; 2) AAV-Vector + IAV group: The empty vector virus was injected beforehand, and 75 PFU PR8 was infused during the challenge experiment as an infection control; 3) AAV-SECTM1 + PBS group: AAV-SECTM1 virus was injected beforehand, and an equal volume of sterile PBS was dripped during the challenge experiment to assess the effect of protein expression on basic physiological indicators; 4) AAV-SECTM1 + IAV group: AAV-SECTM1 virus was injected beforehand, and 75 PFUPR8 was infused during the challenge experiment. This group was the core experimental group.

[0111] Specific steps for virus attack: All operations involving live viruses were performed in a biosafety cabinet in a biosafety level 2 (BSL-2) laboratory, and the procedures were strictly in accordance with biosafety regulations.

[0112] Inhalation anesthesia: Isoflurane inhalation anesthesia was used. Mice were placed in an induction box, and 3%–4% isoflurane gas was introduced. The mice were removed after the righting reflex disappeared and breathing became deep, even, and regular.

[0113] Intranasal infection: Anesthetized mice were placed in a supine position, and a total of 30 μL of IAV virus diluent (containing 75 PFU PR8) was slowly dripped into both nasal cavities using a micropipette. The dripping rate was strictly controlled during the process, and the mice were guided to inhale the virus solution into their lungs through spontaneous respiration.

[0114] Postoperative observation: After the mice have completely inhaled the liquid and there is no obvious choking reaction, they are put back into the cage and placed in a lateral recumbent position until they wake up naturally.

[0115] After infection, the mice were placed in isolation and their physiological state was continuously observed.

[0116] (iv) Monitoring of mouse weight changes and survival status From the day of influenza virus infection, the weight changes of each group of mice were recorded at fixed times every day, and the survival status of the mice was monitored for 14 consecutive days.

[0117] Body weight was recorded as a baseline on the day of infection, and the changes over time were tracked. Simultaneously, the survival of mice in each group during the observation period was assessed to evaluate the effect of recombinant adeno-associated virus-mediated SECTM1 expression on influenza virus infection.

[0118] (v) Detection of viral load in mice On day 6 of influenza virus infection in mice, lung tissue was collected from 5 mice in each group, and tissue RNA was extracted using Trizol (ThermoFisher, #15596026CN). 0.01 g of mouse tissue was weighed, minced in an EP tube, and 200 μL of Trizol was added. The tissue was thoroughly lysed using a sterile, enzyme-free tissue homogenizer. Another 800 μL of Trizol was added, and the mixture was repeatedly inverted and incubated at room temperature for 15 min to ensure complete tissue lysis. Unlysed connective tissue was removed by centrifugation (12000 rpm, 10 min). 200 μL of chloroform was added to the tissue lysis buffer, and the mixture was vigorously vortexed and incubated at room temperature for 10 min. The mixture was then centrifuged at 12000 rpm, 4°C for 20 min, and the supernatant was transferred to a new EP tube. Isopropanol (1.5 times the liquid volume) was added to the EP tube, and the mixture was gently inverted and incubated at room temperature for 10 min. Centrifuge at 12000 rpm, 4℃ for 10 min, discard the supernatant completely, resuspend the precipitate in 75% ethanol, centrifuge at 12000 rpm, 4℃ for 5 min, discard the supernatant. Repeat the ethanol washing step. After aspirating the ethanol and allowing the precipitate to air dry completely at room temperature, dissolve the RNA precipitate in 100 mL of DEPC H2O. The concentration of extracted RNA was determined using NanoDrop (Thermo Fisher, #NanoDrop One), and RNA purity and quality were assessed using 260 / 280 and 260 / 230 indices.

[0119] Using a reverse transcription kit (Akerui, AG11705) and IAV-specific reverse transcription primers, reverse transcription was performed according to the standard procedure of the reverse transcription kit. The cDNA obtained from reverse transcription was also quantified by qRT-PCR using IAV-specific primers (Table 6). The qRT-PCR reaction system was prepared as shown in Table 7, and the reaction procedure was shown in Table 8.

[0120] Table 6 IAV-specific reverse transcription and qRT-PCR primers

[0121] Table 7. Reaction system for qRT-PCR quantification of IAV

[0122] Table 8 Two-step qRT-PCR reaction procedure

[0123] from Figure 9 , Figure 10It can be seen that the mice in the vector group experienced a significant decrease in body weight after infection, with a stable downward trend appearing on the 3rd day post-infection; while the mice in the SECTM1 overexpression group showed a significantly lower rate of body weight loss than the control group, and their lowest average body weight was higher than that of the control group. Regarding survival rate, the mortality rate of mice in the control group was over 50%, while SECTM1 overexpression completely protected against death caused by influenza infection.

[0124] from Figure 11 The results of the relative expression levels of influenza A virus nucleoprotein and viral RNA showed that overexpression of SECTM1 significantly reduced the replication level of viral RNA in the lungs, with an inhibition rate of nearly tenfold, indicating that SECTM1 can significantly reduce IAV replication in vivo.

[0125] The above description is merely an embodiment of the present invention, and the scope of protection of the present invention is not limited to these specific embodiments, but is determined by the claims of the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principle of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of SECTM1 protein in the preparation of drugs for the treatment and / or prevention of influenza virus infection.

2. The application according to claim 1, characterized in that, The influenza viruses include influenza A virus and / or influenza B virus.

3. The application according to claim 1, characterized in that, The SECTM1 protein exerts its anti-influenza virus effect through any of the following mechanisms: A1. As a soluble protein expressed by cells and secreted into the culture supernatant; A2. Recombinant protein added exogenously.

4. Application of the SECTM1 gene in the preparation of gene therapy drugs for the treatment or prevention of influenza virus infection.

5. The application according to claim 4, characterized in that, The gene therapy drug comprises an expression vector encoding a nucleic acid sequence of the SECTM1 protein.

6. The application according to claim 5, characterized in that, The expression vector is an adenovirus vector or a lentivirus vector.

7. A drug for treating or preventing influenza virus infection, characterized in that, The drug contains: B1. An effective amount of SECTM1 protein, and a pharmaceutically acceptable carrier or excipient; or B2. An effective amount of an expression vector containing a nucleic acid sequence encoding the SECTM1 protein, and a pharmaceutically acceptable vector or excipient.

8. The medicament according to claim 7, characterized in that, The SECTM1 protein is a recombinant SECTM1 protein.

9. The drug according to claim 7, characterized in that, The expression vector is an adenovirus vector or a lentivirus vector.

10. A system for screening drugs for the prevention, mitigation, and / or treatment of influenza, characterized in that, The system screens drugs for the prevention, relief, and / or treatment of influenza by determining whether a drug can promote the expression of the SECTM1 gene.

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