Biological material for regulating expression of htra1 gene and application in antiviral therapy

Abstract

The present application belongs to the field of genetic engineering and biological medicine technology, and particularly relates to a biomaterial for regulating HTRA1 gene expression and application thereof in antiviral. Specifically, the present application finds that inhibiting HTRA1 gene can significantly promote replication of H13N2 subtype influenza virus, thereby promoting infection of H13N2 subtype influenza virus, while overexpression of HTRA1 can effectively inhibit replication of H13N2 subtype influenza virus, reduce virus titer, and thus improve the ability to resist influenza virus infection, so that the HTRA1 gene can be used as a key target for regulating replication of H13N2 subtype influenza virus, and thus has good practical application value.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and biomedicine, specifically relating to biomaterials that regulate HTRA1 gene expression and their application in antiviral therapy. Background Technology

[0002] Influenza viruses are significant pathogens that seriously threaten human and animal health, causing seasonal epidemics and even pandemics. Based on differences in viral surface hemagglutinin (HA) and neuraminidase (NA), they can be classified into 18 HA subtypes and 11 NA subtypes. Influenza viruses can also be classified into human influenza viruses, avian influenza viruses, etc., depending on the host they infect. Although antiviral drugs such as neuraminidase inhibitors (e.g., oseltamivir) and polymerase inhibitors (e.g., baloxavir) are used clinically, the high mutation rate of the virus often leads to the emergence of drug-resistant strains, and existing drug targets are mostly concentrated on the virus's own proteins, resulting in relatively simple mechanisms of action. Therefore, exploring the key factors regulating influenza virus infection and replication in host cells and developing novel antiviral strategies based on host-directed therapy (HDT) has become a current research hotspot.

[0003] The HTRA1 gene, short for HtrA serine peptidase 1, is located on human chromosome 10q26.13 and contains a PDZ domain. It is the first member of the human HtrA serine protease family to be discovered, encoding Golgi protease 1 (HtrA1), and primarily functions in the extracellular matrix. By regulating extracellular matrix degradation and remodeling, it participates in various biological processes such as angiogenesis, neuroprotection, and inflammatory responses. However, the inventors found that the specific function of HTRA1 in the host's antiviral innate immune response, particularly during influenza virus infection, has not yet been reported. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention aims to provide biomaterials for regulating HTRA1 gene expression and their application in antiviral therapy. Specifically, this invention has found that inhibiting the HTRA1 gene significantly promotes the replication of H13N2 subtype influenza virus, thereby promoting H13N2 subtype influenza virus infection, while overexpressing HTRA1 effectively inhibits H13N2 subtype influenza virus replication, reduces viral titer, and thus enhances resistance to influenza virus infection. Therefore, the HTRA1 gene can serve as a key target for regulating H13N2 subtype influenza virus replication. Based on the above research findings, this invention has been completed.

[0005] To achieve the above-mentioned technical objectives, the present invention provides the following technical solution:

[0006] In a first aspect, the present invention provides a biomaterial for regulating HTRA1 gene expression, wherein the biomaterial for regulating HTRA1 gene expression includes a biomaterial that inhibits HTRA1 gene expression and a biomaterial that promotes HTRA1 gene expression.

[0007] The biological material used to inhibit HTRA1 gene expression can be short hairpin RNA (shRNA), recombinant lentiviral expression plasmid, recombinant lentiviral expression vector, or host cell that inhibits HTRA1 gene expression.

[0008] The biological material that promotes HTRA1 gene expression can be an expression plasmid that overexpresses the HTRA1 gene and a host cell that overexpresses HTRA1.

[0009] 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.

[0010] The recombinant lentiviral expression plasmid contains the aforementioned shRNA; further, the recombinant lentiviral expression plasmid is obtained by cloning the aforementioned 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.

[0011] The recombinant lentiviral expression vector comprises the aforementioned recombinant lentiviral expression plasmid; further, the recombinant lentiviral expression vector comprises a recombinant lentiviral expression plasmid and an auxiliary plasmid. In this invention, the auxiliary plasmids include psPAX2 and pMD2.G. psPAX2 is a second-generation lentiviral packaging auxiliary plasmid carrying viral gag, pol, rev, and tat genes. It, along with pMD2.G and the aforementioned recombinant lentiviral vector plasmid, constitutes a three-plasmid system, i.e., constructs a recombinant lentiviral expression vector, which is then co-transfected into 293T cells to package recombinant lentivirus, thereby achieving knockdown of the target gene.

[0012] The host cell that inhibits HTRA1 gene expression can be a cell infected with the aforementioned recombinant lentivirus, thereby specifically inhibiting HTRA1 gene expression. In one specific embodiment of the present invention, the cell can be an A549 cell. The recombinant lentivirus is obtained by transfecting 293T cells with the aforementioned recombinant lentivirus expression vector and then packaging them.

[0013] This invention demonstrates through experiments that, compared with the control group, the host cells with inhibited HTRA1 gene expression significantly promote the replication of H13N2 subtype influenza virus and increase the titer of H13N2 subtype influenza virus after infection with H13N2 subtype influenza virus. This can solve the problem of insufficient titer in virus culture and provide high-titer virus raw materials for vaccine production, virology research, etc.

[0014] The expression plasmid for overexpressing the HTRA1 gene can be obtained by ligating the HTRA1 gene to the plasmid; in a specific embodiment of the present invention, the plasmid is pLV4ltr-PGK-ZsGreen(2A)PURO-CMV plasmid.

[0015] The host cells overexpressing HTRA1 can be obtained by transfecting cells with the expression plasmid that overexpresses the HTRA1 gene. In one specific embodiment of the present invention, the cells can be A549 cells.

[0016] Therefore, in a second aspect, the present invention provides the use of the above-described biomaterials regulating HTRA1 gene expression in any one or more of the following:

[0017] (a) Preparation of virus promoters;

[0018] (b) Preparation of viral inhibitors.

[0019] The virus in question is an influenza virus, specifically the H13N2 subtype influenza virus.

[0020] 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. The influenza virus is the H13N2 subtype influenza virus.

[0021] In this invention, the influenza virus promoter can be a common test reagent for non-medical purposes. The common test reagent can be used to promote the proliferation and replication of H13N2 subtype influenza virus, thereby enabling high-titer influenza virus culture. It can be effectively applied to basic research and also provide high-titer virus raw materials for vaccine production.

[0022] The influenza virus inhibitor can be a drug or a general experimental reagent for non-medical purposes. The general experimental reagent can also be used to study the interaction mechanism between the HTRA1 gene and the influenza virus.

[0023] Specifically, the influenza virus inhibitor inhibits the proliferation and replication of the H13N2 subtype influenza virus, thereby significantly inhibiting H13N2 subtype influenza virus infection.

[0024] When the influenza virus inhibitor is a drug, the drug may be an anti-influenza virus infection drug; furthermore, the drug may also include at least one other drug inactive ingredient.

[0025] The inactive components of the drug can be pharmaceutically used carriers, excipients, and diluents. Furthermore, according to conventional methods, it can be formulated into dosage forms such as powders, granules, suspensions, emulsions, syrups, sprays, oral preparations, topical preparations, suppositories, and sterile injectable solutions.

[0026] The non-pharmaceutical active ingredients that may be included, such as carriers, excipients, and diluents, are well known in the art, and those skilled in the art can determine that they meet clinical standards.

[0027] In another specific embodiment of the present invention, the carrier, excipients, and diluents include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil, etc.; no specific limitations are made herein.

[0028] The beneficial technical effects of one or more of the above technical solutions are as follows:

[0029] The aforementioned technical solution reveals for the first time that the HTRA1 gene is a key target regulating the replication of the H13N2 subtype influenza virus. Inhibiting HTRA1 significantly promotes viral replication, thereby increasing viral titer, while overexpressing HTRA1 effectively inhibits viral replication, thereby reducing viral titer, providing a novel mechanism of action for antiviral strategies. Specifically, biomaterials based on HTRA1 inhibition can efficiently enhance the replication efficiency of H13N2 virus in host cells, overcoming the technical bottleneck of insufficient titer in virus culture and providing high-titer viral raw materials for vaccine production and virological research. Furthermore, biomaterials based on HTRA1 overexpression can effectively block H13N2 virus replication, reducing infection rates and providing new candidate targets for the development of anti-influenza drugs, thus possessing significant practical application value. Attached Figure Description

[0030] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0031] Figure 1 This is the sequencing result in Example 2 of the present invention.

[0032] Figure 2This shows the mRNA expression of HTRA1 after it was packaged into lentivirus and then infected A549 in Example 3 of the present invention.

[0033] Figure 3 This shows the mRNA expression of the NP gene of the H13N2 subtype influenza virus after HTRA1 knockdown in Example 4 of the present invention.

[0034] Figure 4 This shows the protein expression of the PB2 gene of H13N2 subtype influenza virus after HTRA1 knockdown in Example 4 of the present invention.

[0035] Figure 5 This illustrates 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.

[0036] Figure 6 This shows the mRNA expression of HTRA1 after A549 was transfected with pLV4ltr-PGK-ZsGreen(2A)PURO-CMV-HTRA1 in Example 6 of this invention.

[0037] Figure 7 This shows the mRNA expression of the NP gene of the H13N2 subtype influenza virus after overexpression of HTRA1 in Example 6 of the present invention.

[0038] Figure 8 This shows the protein expression of the PB2, PB1, and NP genes of the H13N2 subtype influenza virus after overexpression of HTRA1 in Example 6 of the present invention.

[0039] Figure 9 This describes the effect of HTRA1 gene overexpression on the viral titer of H13N2 subtype influenza virus in Example 6 of the present invention. Detailed Implementation

[0040] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0041] It should be noted that the terminology used herein is for descriptive purposes only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. This invention utilizes techniques and methods conventional in the fields of genetic engineering and molecular biology. Those skilled in the art can employ other conventional techniques, methods, and reagents in the art based on the embodiments provided in this invention, without being limited to the specific embodiments of this invention.

[0042] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available. Unless otherwise specified, the experimental methods described are conventional methods in the art.

[0043] Example 1: Design and screening of shRNAs targeting HTRA1

[0044] (1) Design of shRNA targeting HTRA1

[0045] Using the HTRA1 gene as a template (Gene ID: NM_002775.5), the target gene shRNA was designed using the online shRNA design website provided by Thermo Fisher Scientific. The sequences are shown in Table 1.

[0046] Table 1. Sequences corresponding to shRNA

[0047]

[0048] (2) shRNA primer annealing

[0049] shRNA forward and reverse primers were heated in boiling water for 4 minutes, then cooled naturally to room temperature and stored at -20°C for later use.

[0050] Example 2: Construction of a recombinant lentiviral expression plasmid that specifically inhibits HTRA1 gene expression

[0051] (1) pLKO.1-CMV-copGFP-PURO plasmid digestion

[0052] pLKO.1-CMV-copGFP-PURO was double-digested with Age I and EcoRI and incubated at 37 °C for at least 2 hours. The digestion products were recovered using 0.8% agarose gel extraction kit, and the 5 kb band was recovered.

[0053] (2) pLKO.1-CMV-copGFP-PURO shRNA ligation and transformation

[0054] The recovered enzyme digestion products and shRNA were ligated using T4 ligase at 16 °C for 1–5 hours. The ligation products were transformed into DH5α competent cells. After culturing the resulting single-clone colonies for 12 hours, they were sent for sequencing. The sequencing results are as follows: Figure 1 As shown.

[0055] (3) Extraction of target plasmid

[0056] Positive clones were cultured overnight (12-16 hours) in 25-35 mL LB (containing ampicillin), and then the target plasmid was extracted using an endotoxin-free / small-volume extraction kit.

[0057] Example 3 Construction of an A549 cell line that specifically inhibits HTRA1 gene expression

[0058] (1) shRNA knockdown plasmid was transfected into 293T cells to produce lentivirus.

[0059] HEK-293T cells were cultured, and when the cells reached approximately 80% confluency, the knockdown plasmid shRNA-HTRA1 and helper plasmids psPAX2 and pMD2.G were transfected into HEK-293T cells using Lipofectamine™ 3000 at a concentration of 2:2:1. After 24 hours, 5 mL of culture medium was collected (stored at 4°C), and 5–6 mL of medium containing 10% serum-conjugated antibiotics was added.

[0060] Cells and supernatant were collected 48 h after transfection, centrifuged at 15,000 rpm for 3 min, and filtered through a 0.45 µm filter membrane. 8–10 μg / mL polybrene (1000×) was added, and the mixture was stored at -80°C.

[0061] (2) Determination of the optimal concentration of puromycin

[0062] When A549 cells reached 80-90% confluence, puromycin was added for screening. The final concentration of puromycin was initially determined by gradually increasing it in increments of 1 μg from 1 to 10 μg / mL. Then, it was increased in increments of 0.2 μg to pinpoint the exact amount. Cell growth was monitored daily. The optimal concentration of puromycin for screening target cells was the concentration at which cells completely died after 3-5 days of addition. In this experiment, the optimal concentration of puromycin was ultimately determined to be 1.2 μg / mL.

[0063] (3) Lentiviral infection of A549 cells

[0064] A549 cells were seeded in 6-well plates and added with packaged lentivirus (MOI=5) when the cells reached approximately 80% confluence. Depending on the cell condition, the medium could be changed between 24 and 48 hours, and 1.2 μg / mL of puromycin could be added for selection. A stable cell line would form in about one week.

[0065] (4) Identification of A549 cell lines that specifically inhibit HTRA1 gene expression

[0066] Cell sap from stable cell lines was collected, and cellular RNA was extracted according to the SimplyP Total RNA Extraction Kit instructions. After determining the RNA concentration, quantitative real-time PCR was performed using the One Step TB Green® PrimeScript™ RT-PCR Kit II to measure the HTRA1 mRNA level. The results are as follows: Figure 2 As shown, the HTRA1 mRNA expression level in the HTRA1 knockdown A549 cell line (pLKO.1-CMV-copGFP-PURO-shHTRA1-1 / 2 / 3) was significantly lower than that in the control group (shNC), indicating that the HTRA1 knockdown A549 cell line was successfully constructed, and pLKO.1-CMV-copGFP-PURO-shHTRA1-1 / 2 showed better inhibitory effects.

[0067] Example 4: Effect of A549 cell line with specific inhibition of HTRA1 gene expression on the proliferation of H13N2 subtype influenza virus

[0068] (1) A549 cell line that specifically inhibits HTRA1 gene expression by infection with H13N2 subtype influenza virus

[0069] When A549 cells and pLKO.1-CMV-copGFP-PURO-shHTRA1-1 / 2 cells reached approximately 80% confluence, H13N2 subtype influenza virus (0.5 MOI) was inoculated into the cells. After incubation in an incubator for 1 h, F12K medium containing TPCK was added to achieve a final TPCK concentration of 2 μg / mL. After 24 h of culture, viral replication was assessed.

[0070] (2) Virus replication detection

[0071] (2.1) Quantitative PCR detection of viral NP gene expression level

[0072] Cells were collected 24 h after viral infection. Cellular RNA was extracted according to the SimplyP Total RNA Extraction Kit instructions. After determining the RNA concentration, quantitative real-time PCR was performed using the One Step TB Green® PrimeScript™ RT-PCR Kit II to measure the mRNA level of NP. Results are as follows: Figure 3 As shown, the results indicate that the mRNA level of the NP gene 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).

[0073] (2.2) Western blot detection of viral PB2 protein expression level

[0074] Twenty-four hours after H13N2 subtype influenza virus infection, the culture medium was discarded, and the cells were washed three times with PBS. Then, 200 μL of RIPA lysis buffer containing 1% PMSF was added, and the cells were lysed on ice for 40 min. After centrifugation at 12000 g for 5 min at 4°C, the supernatant was collected, and protein concentration was determined. A portion of the sample was subjected to SDS-PAGE electrophoresis, then transferred to a PVDF membrane for Western blot analysis to detect PB2 protein expression. Results are as follows: Figure 4 As shown, the results indicated 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 suggests that inhibiting HTRA1 expression can significantly promote the replication of H13N2 subtype influenza virus.

[0075] (2.3) Detection of the effect of specific inhibition of the HTRA1 gene on the viral titer of H13N2 subtype influenza virus

[0076] Supernatants were collected 24 hours after H13N2 subtype influenza virus infection of A549 cells (shNC) and A549 cell lines specifically inhibiting HTRA1 gene expression (sh HTRA1). The collected supernatants were serially diluted 10-fold (dilution factor 10...). -1 ~10 -10 Cells were inoculated into 96-well plates coated with a monolayer of MDCK cells and cultured for 60 h. Immunofluorescence was then used to detect the effect of specific inhibition of the HTRA1 gene on the viral titer of H13N2 subtype influenza virus. Results are as follows: Figure 5 As shown, the results indicate that specifically inhibiting HTRA1 gene expression can significantly increase the viral titer of H13N2 subtype influenza virus.

[0077] Example 5: Construction of expression plasmid for overexpressing the HTRA1 gene:

[0078] (1) Using the HTRA1 gene (Gene ID: NM_002775.5) as the target gene, a pair of specific primers for amplifying the HTRA1 gene were designed (see Table 2). The HTRA1 gene sequence was amplified by PCR, and the PCR product was recovered by agarose gel electrophoresis.

[0079] Table 2. Specific primers used for amplifying the HTRA1 gene

[0080]

[0081] (2) The pLV4ltr-PGK-ZsGreen(2A)PURO-CMV plasmid was digested with restriction endonucleases Xhol and BamHI. The digestion products were recovered by agarose gel electrophoresis.

[0082] (3) The products recovered in steps (1) and (2) were seamlessly spliced ​​using a seamless cloning kit. 10 μL of the seamless splicing product was transformed into DH5α competent cells. After the single colony was cultured for 12 h, it was sent for sequencing. The results showed that the expression plasmid overexpressing the HTRA1 gene was successfully constructed.

[0083] (4) Incubate the positive clones in 25-35 mL of LB (ampicillin) overnight (12-16 hours later), and then extract the target plasmid using an endotoxin-free / small-volume extraction kit.

[0084] Example 6: Effects of HTRA1 gene overexpression on H13N2 subtype influenza virus

[0085] (1) A549 cells were cultured until they reached approximately 80% confluency. The expression plasmid overexpressing the HTRA1 gene was then transfected into A549 cells (OE-HTRA1) using Lipofectamine™ 3000. Cells were collected 24 h after transfection and centrifuged at 3000 rpm for 3 min. RNA was extracted from the processed cell solution according to the SimplyP Total RNA Extraction Kit instructions. After determining the RNA concentration, quantitative real-time PCR was performed using the One Step TB Green® PrimeScript™ RT-PCR Kit II to measure the HTRA1 mRNA level. The results are as follows: Figure 6 As shown, the results indicate that HTRA1 is overexpressed in A549 cells (OE-HTRA1).

[0086] (2) The A549 cells from the OE-NC group and the A549 cells from the OE-HTRA1 group obtained in step (1) were infected with H13N2 influenza virus (MOI=0.01). After 24 h of infection, the cell fluid was collected, and cellular RNA was extracted according to the instructions of the SimplyP Total RNA Extraction Kit. After determining the RNA concentration, quantitative real-time PCR was performed using the One Step TB Green® PrimeScript™ RT-PCR Kit II to detect the mRNA expression of the NP gene of H13N2 influenza virus. The results are as follows: Figure 7 As shown, the results indicate that overexpression of HTRA1 inhibited the replication of the H13N2 influenza virus.

[0087] (3) Simultaneously, cells were lysed and the supernatant was collected for SDS-PAGE electrophoresis, then transferred to a PVDF membrane for Western blot detection of the expression of H13N2 influenza virus PB1, PB2, and NP proteins. Results are as follows: Figure 8 As shown, the results indicate that overexpression of HTRA1 inhibited the replication of the H13N2 influenza virus.

[0088] (4) Supernatants were collected 24 h after infection of A549 cells (OE-NC) and A549 cells overexpressing HTRA1 (OE-HTRA1) with H13N2 subtype influenza virus. The collected supernatants were serially diluted 10-fold (dilutions were 10... -1 ~10 -10 The cells were inoculated into 96-well plates coated with MDCK monolayers and cultured for 60 h. The viral titers of H13N2 subtype influenza virus in different cell lines were then detected by immunofluorescence. Results are as follows: Figure 9 As shown, the results indicate that overexpression of HTRA1 can significantly reduce the viral titer of H13N2 subtype influenza virus.

[0089] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of a biomaterial that regulates HTRA1 gene expression in any one or more of the following: (a) Preparation of virus promoters; (b) Preparation of viral inhibitors; in, The virus is an H13N2 subtype influenza virus, and the biological material regulating HTRA1 gene expression includes biological materials that inhibit HTRA1 gene expression and biological materials that promote HTRA1 gene expression; the biological material that inhibits HTRA1 gene expression is used to prepare an influenza virus promoter; the biological material that promotes HTRA1 gene expression is used to prepare an influenza virus inhibitor; wherein, the influenza virus is an H13N2 subtype influenza virus. The biological material used to inhibit HTRA1 gene expression is shRNA, recombinant lentiviral expression plasmid, recombinant lentiviral expression vector, or host cell that inhibits HTRA1 gene expression. The biological materials used to promote HTRA1 gene expression are expression plasmids that overexpress the HTRA1 gene and host cells that overexpress HTRA1.

2. The application as described in claim 1, characterized in that, 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.

3. The application as described in claim 1, characterized in that, The recombinant lentiviral expression plasmid contains the shRNA.

4. The application as described in claim 1, characterized in that, The recombinant lentiviral expression vector comprises the recombinant lentiviral expression plasmid and helper plasmids; the helper plasmids include psPAX2 and pMD2.G.

5. The application as described in claim 1, characterized in that, The host cell that inhibits HTRA1 gene expression is a cell that is specifically inhibited by infection with a recombinant lentivirus; the cell is an A549 cell; the recombinant lentivirus is obtained by transfecting 293T cells with the recombinant lentivirus expression vector and then packaging it.

6. The application as described in claim 1, characterized in that, The expression plasmid for overexpressing the HTRA1 gene was obtained by ligating the HTRA1 gene to the plasmid; the plasmid was pLV4ltr-PGK-ZsGreen(2A)PURO-CMV plasmid.

7. The application as described in claim 1, characterized in that, The host cell for overexpressing HTRA1 was obtained by transfecting cells with the expression plasmid that overexpresses the HTRA1 gene; the cell was A549 cell.

8. The application as described in claim 1, characterized in that, The influenza virus promoter is a common test reagent for non-medical use, and it is used to promote the proliferation and replication of the H13N2 subtype influenza virus. The influenza virus inhibitor is a drug or a common test reagent for non-medical purposes; the influenza virus inhibitor is used to inhibit the proliferation and replication of the H13N2 subtype influenza virus, thereby inhibiting the infection of the H13N2 subtype influenza virus.

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