Biological material for regulating and controlling HTRA1 gene expression and application of biological material in virus resistance

Abstract

The invention belongs to the technical field of gene engineering and biological medicine, and particularly relates to a biological material for regulating HTRA1 gene expression and application of the biological material in virus resistance. Specifically, the research finds that the replication of the H13N2 subtype influenza virus can be obviously promoted by inhibiting the HTRA1 gene so as to promote the infection of the H13N2 subtype influenza virus, and the replication of the H13N2 subtype influenza virus can be effectively inhibited by overexpressing the HTRA1, so that the virus titer is reduced, and the anti-influenza virus infection capability is improved; therefore, the HTRA1 gene can be used as a key target spot for regulating and controlling replication of the H13N2 subtype influenza virus, so that the HTRA1 gene has a good practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical fields of genetic engineering and biomedicine, and particularly relates to a biomaterial for regulating HTRA1 gene expression and its application in antiviral treatment. Background Art

[0002] Influenza viruses are serious pathogens that threaten human and animal health, causing seasonal epidemics and even pandemics. They are classified into 18 HA subtypes and 11 NA subtypes based on differences in the viral surface hemagglutinin (HA) and neuraminidase (NA). Influenza viruses are further categorized into human and avian influenza viruses, depending on the host they infect. Although antiviral drugs such as neuraminidase inhibitors (such as oseltamivir) and polymerase inhibitors (such as baloxavir) are currently in clinical use, the high mutation rate of the virus often leads to the emergence of drug-resistant strains. Furthermore, existing drug targets mostly target viral proteins, resulting in relatively limited mechanisms of action. Therefore, exploring key factors regulating influenza virus infection and replication in host cells and developing novel antiviral strategies based on host-directed therapy (HDT) have become current research hotspots.

[0003] The HTRA1 gene, fully named HtrA serine peptidase 1, is located at human chromosome 10q26.13. It possesses a PDZ domain and is the first member of the human HtrA serine protease family to be discovered. It encodes Golgi protease 1 (HtrA1), which primarily functions within the extracellular matrix. By regulating extracellular matrix degradation and remodeling, it participates in various biological processes, including angiogenesis, neuroprotection, and inflammatory responses. However, the inventors discovered that the specific function of HTRA1 in the host's antiviral innate immune response, particularly during influenza virus infection, has yet to be fully understood. Summary of the Invention

[0004] To address the shortcomings of the prior art, the present invention aims to provide biomaterials that regulate HTRA1 gene expression and their use in antiviral treatments. Specifically, the present invention has discovered that inhibiting the HTRA1 gene significantly promotes the replication of the H13N2 subtype influenza virus, thereby facilitating infection. Meanwhile, overexpressing HTRA1 can effectively inhibit the replication of the H13N2 subtype influenza virus, reducing viral titers and thereby improving resistance to influenza virus infection. Therefore, the HTRA1 gene can serve as a key target for regulating the replication of the H13N2 subtype influenza virus. Based on these research findings, the present invention was completed.

[0005] In order to achieve the above technical objectives, the technical solutions provided by the present invention are as follows: The first aspect of the present invention provides a biomaterial for regulating HTRA1 gene expression, wherein the biomaterial for regulating HTRA1 gene expression includes a biomaterial for inhibiting HTRA1 gene expression and a biomaterial for promoting HTRA1 gene expression; Wherein, the biological material that inhibits HTRA1 gene expression can be short hairpin RNA (shRNA), a recombinant lentiviral expression plasmid, a recombinant lentiviral expression vector or a host cell that inhibits HTRA1 gene expression; The biological material that promotes the expression of the HTRA1 gene can be an expression plasmid that overexpresses the HTRA1 gene and a host cell that overexpresses HTRA1.

[0006] The shRNA comprises a sense strand and an antisense strand, wherein the sense strand has a nucleotide sequence as shown in any one of SEQ ID NO.1, SEQ ID NO.3 or SEQ ID NO.5, and the antisense strand has a nucleotide sequence as shown in any one of SEQ ID NO.2, SEQ ID NO.4 or SEQ ID NO.6.

[0007] The recombinant lentiviral expression plasmid comprises the above-mentioned shRNA; further, the recombinant lentiviral expression plasmid is obtained by cloning the above-mentioned shRNA into a lentiviral plasmid vector. In a specific embodiment of the present invention, the lentiviral plasmid vector can be a pLKO.1 plasmid vector.

[0008] The recombinant lentiviral expression vector comprises the aforementioned recombinant lentiviral expression plasmid; further, the recombinant lentiviral expression vector comprises the recombinant lentiviral expression plasmid and a helper plasmid. In the present invention, the helper plasmids include psPAX2 and pMD2.G. psPAX2 is a second-generation lentiviral packaging helper plasmid carrying the viral gag, pol, rev, and tat genes. Together with pMD2.G and the aforementioned recombinant lentiviral vector plasmid, it forms a three-plasmid system, constructing the recombinant lentiviral expression vector. This system is then co-transfected into cells such as 293T cells to package the recombinant lentivirus, thereby achieving knockdown of the target gene.

[0009] The host cells that inhibit HTRA1 gene expression can be cells infected with the aforementioned recombinant lentivirus, thereby specifically inhibiting HTRA1 gene expression. In one embodiment of the present invention, the cells can be A549 cells. The recombinant lentivirus is obtained by transfecting 293T cells with the aforementioned recombinant lentiviral expression vector and packaging it.

[0010] The present invention has demonstrated through experiments that, compared with a control group, the host cells in which HTRA1 gene expression is inhibited can significantly promote the replication of H13N2 subtype influenza virus and increase the titer of H13N2 subtype influenza virus after being infected with H13N2 subtype influenza virus, thereby solving the problem of insufficient titer in virus culture and providing high-titer virus raw materials for vaccine production, virology research, etc.

[0011] The expression plasmid for overexpressing the HTRA1 gene can be obtained by connecting the HTRA1 gene with a plasmid; in a specific embodiment of the present invention, the plasmid is the pLV4ltr-PGK-ZsGreen (2A) PURO-CMV plasmid.

[0012] The host cell overexpressing HTRA1 can be obtained by transfecting the above-mentioned expression plasmid overexpressing HTRA1 gene into the cell. In a specific embodiment of the present invention, the cell can be A549 cell.

[0013] Therefore, the second aspect of the present invention provides the use of the above-mentioned biomaterial for regulating HTRA1 gene expression in any one or more of the following: (a) preparing a virus promoter; (b) Preparation of viral inhibitors.

[0014] Wherein, the virus is an influenza virus, further an H13N2 subtype influenza virus.

[0015] Furthermore, the biomaterial that inhibits HTRA1 gene expression is used to prepare an influenza virus promoter. The biomaterial that promotes HTRA1 gene expression is used to prepare an influenza virus inhibitor. Wherein, the influenza virus is an H13N2 subtype influenza virus.

[0016] In the present invention, the influenza virus promoter can be a common test reagent for non-medical use. The common test reagent can be used to promote the proliferation and replication of H13N2 subtype influenza virus, thereby being used for high-titer cultivation of influenza virus. It can be effectively applied to basic research and also provide high-titer virus raw materials for vaccine production, etc.

[0017] The influenza virus inhibitor may be a drug or a common test reagent for non-medical use. The common test reagent may also be used to study the interaction mechanism between the HTRA1 gene and the influenza virus.

[0018] Specifically, the influenza virus inhibitor specifically inhibits the proliferation and replication of H13N2 subtype influenza virus, thereby significantly inhibiting the infection of H13N2 subtype influenza virus.

[0019] When the influenza virus inhibitor is a drug, the drug may be a drug for resisting influenza virus infection; further, the drug may further include at least one other inactive drug ingredient.

[0020] The inactive ingredients of the drug can be carriers, excipients, diluents, etc. commonly used in pharmacy. Moreover, according to common methods, the drug can be prepared into oral dosage forms such as powders, granules, suspensions, emulsions, syrups, sprays, external preparations, suppositories, and sterile injection solutions.

[0021] The non-drug active ingredients such as carriers, excipients and diluents that may be included are well known in the art, and those skilled in the art can determine whether they meet clinical standards.

[0022] In another specific embodiment of the present invention, the carrier, excipient and diluent include but are not limited to lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, etc.; no specific limitation is made here.

[0023] Beneficial technical effects of one or more of the above technical solutions: The above technical solution reveals for the first time that the HTRA1 gene is a key target for regulating the replication of the H13N2 subtype influenza virus. Inhibiting HTRA1 can significantly promote viral replication, thereby increasing viral titers, while overexpressing HTRA1 can effectively inhibit viral replication, thereby reducing viral titers, providing a new mechanism of action for antiviral strategies. Specifically, biomaterials based on inhibiting HTRA1 can effectively enhance the replication efficiency of the H13N2 virus in host cells, resolving the technical bottleneck of insufficient titers in virus culture and providing high-titer viral raw materials for vaccine production and virological research. Biomaterials based on overexpressing HTRA1 can effectively block H13N2 virus replication and reduce infection rates, providing new candidate targets for the development of anti-influenza drugs, and therefore have excellent practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0025] Figure 1 This is the sequencing result in Example 2 of the present invention.

[0026] Figure 2This is the mRNA expression of HTRA1 after shHTRA1 was packaged into lentivirus and infected into A549 cells in Example 3 of the present invention.

[0027] Figure 3 This is the mRNA expression of the H13N2 subtype influenza virus NP gene after knocking down HTRA1 in Example 4 of the present invention.

[0028] Figure 4 This is the protein expression of the PB2 gene of the H13N2 subtype influenza virus after knocking down HTRA1 in Example 4 of the present invention.

[0029] Figure 5 This is the effect of specifically inhibiting the HTRA1 gene on the viral titer of the H13N2 subtype influenza virus in Example 4 of the present invention.

[0030] Figure 6 This is the mRNA expression of HTRA1 after A549 cells were transfected with pLV4ltr-PGK-ZsGreen(2A)PURO-CMV-HTRA1 in Example 6 of the present invention.

[0031] Figure 7 This is the mRNA expression of the H13N2 subtype influenza virus NP gene after overexpression of HTRA1 in Example 6 of the present invention.

[0032] Figure 8 This is the protein expression of PB2, PB1, and NP genes of H13N2 subtype influenza virus after overexpression of HTRA1 in Example 6 of the present invention.

[0033] Figure 9 This is the effect of overexpressing the HTRA1 gene on the viral titer of the H13N2 subtype influenza virus in Example 6 of the present invention. DETAILED DESCRIPTION

[0034] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0035] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should also be understood that when the terms "comprising" and / or "including" are used in this manual, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof. The present invention has used conventional techniques and methods in the field of genetic engineering and molecular biology. Those skilled in the art can adopt other conventional techniques, methods and reagents in this area on the basis of the embodiments provided by the present invention, without being limited to the limitation of the specific embodiments of the present invention.

[0036] The present invention is described in detail below with reference to the accompanying drawings and specific examples. In the following examples, the materials and reagents used were obtained from commercial sources unless otherwise specified. The experimental methods described are conventional methods in the art unless otherwise specified.

[0037] Example 1 Design and screening of shRNA targeting HTRA1 (1) Design of shRNA targeting HTRA1 Using the HTRA1 gene as a template (Gene ID: NM_002775.5), the shRNA of the target gene was designed using the online shRNA design website provided by Thermo Fisher Scientific. The sequences are shown in Table 1.

[0038] Table 1 Sequences corresponding to shRNA

[0039] (2) shRNA primer annealing The shRNA forward and reverse strand primers were heated in boiling water for 4 minutes, cooled naturally to room temperature, and then stored at -20°C for later use.

[0040] Example 2 Construction of a recombinant lentiviral expression plasmid that specifically inhibits HTRA1 gene expression (1) Enzyme digestion of pLKO.1-CMV-copGFP-PURO plasmid pLKO.1-CMV-copGFP-PURO was double-digested with Age I and EcoR I and incubated at 37°C for at least 2 hours. The digestion product was recovered using 0.8% agarose gel extraction kit, and a 5 kb band was recovered.

[0041] (2) pLKO.1-CMV-copGFP-PURO shRNA ligation and transformation The recovered enzyme digestion product and shRNA were ligated with T4 ligase at 16°C for 1-5 hours. The ligation product was transformed into DH5α competent cells, and the monoclonal colonies grown were cultured for 12 hours and then sent for sequencing. The sequencing results were as follows: Figure 1 shown.

[0042] (3) Extraction of target plasmid Culture the positive clones in 25-35 mL LB (containing ampicillin) overnight (12-16 hours), and then extract the target plasmid using an endotoxin removal / mini-prep kit.

[0043] Example 3 Construction of A549 cell line specifically inhibiting HTRA1 gene expression (1) Transfection of shRNA knockdown plasmid into 293T cells to produce lentivirus Culture HEK-293T cells. When the cells reach approximately 80% confluency, transfect the HEK-293T cells with the knockdown plasmid shRNA-HTRA1 and the helper plasmids psPAX2 and pMD2.G at a ratio of 2:2:1 using Lipofectamine™ 3000. After 24 hours, collect 5 mL of culture medium (store at 4°C) and add 5-6 mL of culture medium containing 10% serum and double-stranded antibody.

[0044] 48 h after transfection, cells and supernatant were collected, centrifuged at 15,000 rpm for 3 min, filtered through a 0.45 µm filter, and 8–10 μg / mL polybrene (1000×) was added. Store at -80°C.

[0045] (2) Determination of the optimal puromycin concentration When A549 cells reach 80-90% confluency, puromycin is added for selection. The final puromycin concentration is initially increased in 1-μg increments between 1 and 10 μg / mL. Once the approximate range is established, the final puromycin concentration is then increased in 0.2 μg increments to determine the precise dosage. Cell growth should be monitored daily. The optimal concentration for selecting target cells is the concentration that results in complete cell death 3-5 days after addition of puromycin. In this experiment, the optimal puromycin concentration was determined to be 1.2 μg / mL.

[0046] (3) Lentivirus infection of A549 cells A549 cells were plated in a 6-well plate. When cells reached approximately 80% confluence, the packaged lentivirus (MOI = 5) was added. Depending on the cell status, the medium was changed every 24-48 hours, and 1.2 μg / mL of puromycin was added for selection. A stable cell line should be established in approximately one week.

[0047] (4) Identification of A549 cell lines that specifically inhibit HTRA1 gene expression The cell fluid of the stable cell line was collected and the cellular RNA was extracted according to the instructions of the SimplyP total RNA extraction kit. After measuring the RNA concentration, the One Step TB Green® PrimeScript™ RT-PCR Kit II was used for fluorescence quantitative PCR to measure the mRNA level of HTRA1. The results are shown in Figure 2. Figure 2 As shown in the figure, the HTRA1 mRNA expression level of the HTRA1 knockdown A549 cell line (pLKO.1-CMV-copGFP-PURO-shHTRA1-1 / 2 / 3) was significantly lower than that of the control group (shNC), indicating that the HTRA1 knockdown A549 cell line was successfully constructed, among which pLKO.1-CMV-copGFP-PURO-shHTRA1-1 / 2 had a better inhibitory effect.

[0048] Example 4 Effect of A549 Cell Line with Specific Inhibition of HTRA1 Gene Expression on the Proliferation of H13N2 Subtype Influenza Virus (1) Infection of H13N2 subtype influenza virus in A549 cell line specifically inhibits HTRA1 gene expression A549 cells and pLKO.1-CMV-copGFP-PURO-shHTRA1-1 / 2 cells were cultured to approximately 80% confluence. H13N2 subtype influenza virus (MOI) was inoculated into the cells at 0.5 MOI. After incubation for 1 hour, F12K medium containing TPCK was added to a final TPCK concentration of 2 μg / mL. Viral replication was assessed after 24 hours of culture.

[0049] (2) Virus replication detection (2.1) Fluorescence quantitative detection of viral NP gene expression level 24 hours after virus infection, cells were collected and cellular RNA was extracted according to the instructions of the SimplyP total RNA extraction kit. After measuring the RNA concentration, fluorescent quantitative PCR was performed using the One Step TB Green® PrimeScript™ RT-PCR Kit II to measure the mRNA level of NP. Figure 3 As shown, the results showed that the mRNA level of NP gene in A549-sh HTRA1-1 / 2 cells infected with H13N2 subtype influenza virus was significantly higher than that in A549 cell control group (shNC).

[0050] (2.2) Western blot detection of viral PB2 protein expression level After 24 hours of infection with H13N2 subtype influenza virus, the cells were aspirated and the medium was discarded. After washing three times with PBS, 200 μL of RIPA lysis buffer containing 1% PMSF was added and the cells were lysed on ice for 40 minutes. The cells were centrifuged at 12,000 g for 5 minutes at 4°C. The supernatant was collected and the protein concentration was determined. A portion of the sample was subjected to SDS-PAGE electrophoresis and then transferred to a PVDF membrane for Western blot analysis of PB2 protein expression. The results are shown in Figure 2. Figure 4 As shown, the results showed that the amount of PB2 protein in A549-sh HTRA1-1 / 2 cells infected with H13N2 subtype influenza virus was significantly higher than that in the A549 cell control group (shNC). This result indicates that inhibiting HTRA1 expression can significantly promote the replication of H13N2 subtype influenza virus.

[0051] (2.3) Detecting the effect of specific inhibition of the HTRA1 gene on the viral titer of H13N2 subtype influenza virus A549 cells (shNC) and A549 cell line with specific inhibition of HTRA1 gene expression (shHTRA1) were infected with H13N2 subtype influenza virus for 24 hours and the supernatant was collected. The collected supernatant was serially diluted 10-fold (dilutions were 10 -1 ~10 -10 ) were inoculated into 96-well plates with MDCK monolayer cells, and the effect of specific inhibition of the HTRA1 gene on the titer of H13N2 subtype influenza virus was detected by immunofluorescence after 60 h of culture. Figure 5 As shown, the results showed that specific inhibition of HTRA1 gene expression can significantly increase the viral titer of H13N2 subtype influenza virus.

[0052] Example 5 Construction of an expression plasmid for overexpressing the HTRA1 gene: (1) Using the HTRA1 gene (Gene ID: NM_002775.5) as the target gene, a pair of specific primers were designed for amplifying the HTRA1 gene (see Table 2). The HTRA1 gene sequence was amplified by PCR, and the PCR product was recovered after agarose gel electrophoresis.

[0053] Table 2 Specific primers used to amplify the HTRA1 gene

[0054] (2) The pLV4ltr-PGK-ZsGreen(2A)PURO-CMV plasmid was double-digested with restriction endonucleases Xhol and BamHI, and the digestion products were recovered by agarose gel electrophoresis.

[0055] (3) The products recovered in step (1) and step (2) were seamlessly spliced ​​using a seamless cloning kit. 10 μL of the seamless splicing product was transformed into DH5α competent cells, and the resulting monoclonal colonies were cultured for 12 hours and then sent for sequencing. The results showed that the expression plasmid overexpressing the HTRA1 gene was successfully constructed.

[0056] (4) Culture the positive clones in 25-35 mL LB (ampicillin) overnight (after 12-16 hours), and then use the endotoxin removal / mini-prep kit to extract the target plasmid.

[0057] Example 6 Effect of Overexpression of HTRA1 Gene on H13N2 Subtype Influenza Virus (1) Culture A549 cells. When the cells grow to about 80%, transfect the A549 cells with the expression plasmid that overexpresses the HTRA1 gene using LipofectamineTM3000 (OE-HTRA1). 24 hours after transfection, collect the cells and centrifuge at 3000 rpm for 3 minutes. Extract the cellular RNA from the treated cell fluid according to the instructions of the SimplyP total RNA extraction kit. After determining the RNA concentration, use One Step TB Green® PrimeScript™ RT-PCR Kit II for fluorescence quantitative PCR to determine the mRNA level of HTRA1. The results are as follows: Figure 6 As shown, the results demonstrated that HTRA1 was overexpressed in A549 cells (OE-HTRA1).

[0058] (2) The A549 cells in the OE-NC group and the A549 cells in the OE-HTRA1 group obtained in step (1) were infected with H13N2 influenza virus (MOI = 0.01). After 24 hours of virus infection, the cell fluid was collected and the cellular RNA was extracted according to the instructions of the SimplyP total RNA extraction kit. After determining the RNA concentration, the One Step TB Green® PrimeScript™ RT-PCR Kit II was used for fluorescence quantitative PCR to detect the mRNA expression of the H13N2 influenza virus NP gene. The results are shown in Figure 2. Figure 7 As shown, the results showed that overexpression of HTRA1 inhibited the replication of H13N2 influenza virus.

[0059] (3) At the same time, the cells were lysed and the supernatant was collected for SDS-PAGE electrophoresis, then transferred to a PVDF membrane and Western blot was performed to detect the expression of H13N2 influenza virus PB1, PB2 and NP proteins. The results are as follows Figure 8 As shown, the results showed that overexpression of HTRA1 inhibited the replication of H13N2 influenza virus.

[0060] (4) After 24 hours of infection of A549 cells (OE-NC) and A549 cells overexpressing HTRA1 (OE-HTRA1) with H13N2 subtype influenza virus, the supernatants were collected. The collected supernatants were diluted 10-fold (dilutions were 10 -1 ~10 -10 ) were inoculated into 96-well plates with MDCK monolayer cells, and the virus titers of H13N2 subtype influenza virus in different cell lines were detected by immunofluorescence after 60 h of culture. Figure 9 As shown, the results showed that overexpression of HTRA1 could significantly reduce the viral titer of H13N2 subtype influenza virus.

[0061] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A biomaterial for regulating HTRA1 gene expression, characterized in that: The biological materials for regulating HTRA1 gene expression include biological materials that inhibit HTRA1 gene expression and biological materials that promote HTRA1 gene expression; Wherein, the biological material for inhibiting HTRA1 gene expression is shRNA, recombinant lentiviral expression plasmid, recombinant lentiviral expression vector or host cell for inhibiting HTRA1 gene expression; The biological materials for promoting the expression of the HTRA1 gene are an expression plasmid for overexpressing the HTRA1 gene and a host cell for overexpressing the HTRA1 gene.

2. The biomaterial according to claim 1, wherein The shRNA includes a sense strand and an antisense strand, wherein the sense strand has a nucleotide sequence as shown in any one of SEQ ID NO.1, SEQ ID NO.3 or SEQ ID NO.5, and the antisense strand has a nucleotide sequence as shown in any one of SEQ ID NO.2, SEQ ID NO.4 or SEQ ID NO.

6.

3. The biomaterial according to claim 1, wherein The recombinant lentiviral expression plasmid contains the shRNA.

4. The biomaterial according to claim 1, wherein The recombinant lentiviral expression vector comprises the recombinant lentiviral expression plasmid and an auxiliary plasmid; the auxiliary plasmid includes psPAX2 and pMD2.G.

5. The biomaterial according to claim 1, wherein The host cells that inhibit HTRA1 gene expression are cells infected by recombinant lentiviruses, thereby specifically inhibiting HTRA1 gene expression; the cells are A549 cells; and the recombinant lentivirus is obtained by transfecting 293T cells with the recombinant lentivirus expression vector and packaging the result.

6. The biomaterial according to claim 1, wherein The expression plasmid for overexpressing the HTRA1 gene is obtained by connecting the HTRA1 gene with a plasmid; the plasmid is the pLV4ltr-PGK-ZsGreen (2A) PURO-CMV plasmid.

7. The biomaterial according to claim 1, wherein The host cell overexpressing HTRA1 is obtained by transfecting the expression plasmid overexpressing the HTRA1 gene into the cell; the cell is A549 cell.

8. Use of the biomaterial for regulating HTRA1 gene expression according to any one of claims 1 to 7 in any one or more of the following: (a) preparing a virus promoter; (b) preparing viral inhibitors; in, The virus is an H13N2 subtype influenza virus.

9. The use according to claim 8, characterized in that The biological material for inhibiting HTRA1 gene expression is used to prepare an influenza virus promoter; the biological material for promoting HTRA1 gene expression is used to prepare an influenza virus inhibitor; wherein the influenza virus is an H13N2 subtype influenza virus.

10. The use according to claim 9, characterized in that The influenza virus promoter is a common test reagent for non-medical use, and is used to promote the proliferation and replication of H13N2 subtype influenza virus; The influenza virus inhibitor is a common test reagent for medicine or non-medical use; the influenza virus inhibitor is used to inhibit the proliferation and replication of H13N2 subtype influenza virus, thereby inhibiting the infection of H13N2 subtype influenza virus.

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