Biological material for inhibiting expression of ide gene and application in anti-influenza virus

Abstract

The present application belongs to the field of genetic engineering and biological medicine technology, and particularly relates to a biomaterial for inhibiting expression of an IDE gene and application thereof in resisting influenza viruses. The present application finds that specific inhibition of expression of the IDE gene can significantly inhibit infection of influenza viruses, particularly H13N2 subtype influenza viruses. Specifically, the present application finds that the mRNA expression level of the NP gene of the H13N2 subtype influenza virus and the expression level of the PB2 protein in an A549 cell line with specific inhibition of expression of the IDE gene are significantly reduced; meanwhile, the virus titer after the H13N2 subtype influenza virus infects the A549 cell line with specific inhibition of expression of the IDE gene is also significantly lower than that of a control group. The present application proves that specific inhibition of expression of the IDE gene can significantly inhibit infection of the H13N2 subtype influenza virus, and provides a new idea for resisting influenza virus infection, and therefore 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 inhibit IDE gene expression and their application in anti-influenza virus therapy. Background Technology

[0002] Influenza virus is a segmented RNA virus, classified into 18 HA subtypes and 11 NA subtypes based on differences in viral surface hemagglutinin (HA) and neuraminidase (NA). Influenza viruses can also be classified into human influenza viruses, avian influenza viruses, etc., depending on the host they infect. Host factors play a crucial role in influenza virus infection and replication; therefore, studying the interaction between host factors and influenza virus is of great significance for the prevention and control of this virus.

[0003] Insulin-degrading enzyme (IDE) is an enzyme widely expressed in various tissues of the human body, playing a crucial role, especially in the brain. Its main function is to degrade insulin and Aβ proteins, maintaining a balance between insulin and Aβ, protecting nerve cells from damage, and thus ensuring normal cognitive function. In patients with Alzheimer's disease and diabetes, IDE activity is often inhibited, leading to the accumulation of Aβ in the brain and impaired insulin signaling. This imbalance not only exacerbates insulin resistance but also intensifies Aβ deposition in the brain, triggering synaptic damage and neuronal dysfunction, ultimately leading to cognitive decline. However, the inventors have found that there are currently no reports of IDE being used to combat influenza viruses. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide biomaterials that inhibit IDE gene expression and their application in combating influenza viruses. Specifically, the present invention has found that specifically inhibiting IDE gene expression can significantly suppress influenza virus infection, particularly the H13N2 subtype influenza virus. Based on the above research findings, the present 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 biological material for inhibiting the expression of the IDE gene, wherein the biological material may be short hairpin RNA (shRNA), recombinant lentiviral expression plasmid, recombinant lentiviral expression vector or host cell.

[0007] The shRNA comprises a sense strand and an antisense strand, wherein the sense strand has a nucleotide sequence as shown in SEQ ID NO.1; the corresponding antisense strand has a nucleotide sequence as shown in SEQ ID NO.2; or, the sense strand has a nucleotide sequence as shown in SEQ ID NO.3, and the corresponding antisense strand has a nucleotide sequence as shown in SEQ ID NO.4.

[0008] Furthermore, the sense strand of the shRNA has a nucleotide sequence as shown in SEQ ID NO.1, and the antisense strand has a nucleotide sequence as shown in SEQ ID NO.2.

[0009] 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.

[0010] 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.

[0011] The host cell can be a cell that has been infected with a recombinant lentivirus, thereby specifically inhibiting the expression of the IDE gene. In one specific embodiment of the present invention, the host cell can be an A549 cell. The recombinant lentivirus is obtained by transfecting 293T cells with the above-mentioned recombinant lentivirus expression vector and then packaging them.

[0012] This invention demonstrates through experiments that, compared with the control group, the above-mentioned host cells, after being infected by the H13N2 subtype influenza virus, can significantly inhibit the replication of the H13N2 subtype influenza virus and reduce the titer of the H13N2 subtype influenza virus, and therefore can be used as an anti-influenza virus product.

[0013] Therefore, in a second aspect, the present invention provides the use of the above-described biomaterials that inhibit IDE gene expression in the preparation of anti-influenza virus products.

[0014] In this invention, the influenza virus is further defined as the H13N2 subtype influenza virus.

[0015] The anti-influenza virus product can be a drug or a general testing reagent for non-medical purposes. The general testing reagent can be used for basic research on the relationship between the IDE gene and the influenza virus.

[0016] Specifically, the anti-influenza virus product inhibits the proliferation and replication of the H13N2 subtype influenza virus, thereby significantly inhibiting the infection of the H13N2 subtype influenza virus.

[0017] When the anti-influenza virus product is a drug, the drug can be a drug for treating influenza virus infection; furthermore, the drug also contains pharmaceutically acceptable excipients and / or carriers, and the content of the excipients and / or carriers in the drug can be 1% by weight to 98% by weight, including but not limited to 5% by weight, 10% by weight, 20% by weight, 30% by weight, 40% by weight, 50% by weight, 60% by weight, 70% by weight, 80% by weight, 90% by weight, 95% by weight, and 98% by weight, without specific limitations.

[0018] The dosage form of the traditional Chinese medicine preparation of this invention is prepared using conventional methods to produce a pharmaceutically acceptable conventional liquid or solid dosage form. Specifically, the dosage form of the drug can be oral tablets, oral liquids, syrups, granules, capsules, tablets, or pills. This makes it easy to carry and take, which is beneficial for clinical promotion and practical application.

[0019] To enable the above dosage forms to be realized, pharmaceutically acceptable excipients need to be added during the preparation of these dosage forms, such as: ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffering substances such as phosphates, glycerol, sorbitol, potassium sorbate, a mixture of partial glycerides of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, magnesium trisilicate, polyvinylpyrrolidone, etc.

[0020] Oral tablets and capsules may contain excipients such as binders, like syrups, gum arabic, sorbitol, astragalus gum, or polyvinylpyrrolidone; fillers such as lactose, sucrose, corn starch, calcium phosphate, sorbitol, glycine; lubricants such as magnesium stearate, talc, polyethylene glycol, silica; disintegrants such as potato starch; or acceptable wetting agents such as sodium lauryl sulfate. Oral tablets may be coated using pharmaceutically known methods.

[0021] Oral liquids can be formulated as water and oil suspensions, solutions, emulsions, syrups, etc., or as dry products, to be replenished with water or other suitable media before use. These liquid formulations may contain conventional additives such as suspending agents, sorbitol, cellulose methyl ether, glucose syrup, gelling agents, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel, hydrogenated edible oils, emulsifiers such as lecithin, sorbitan monooleate, gum arabic; or non-aqueous carriers including almond oil, oils such as glycerin, ethylene glycol, or ethanol; and preservatives including methylparaben, propylparaben, or sorbic acid.

[0022] The beneficial technical effects of one or more of the above technical solutions are as follows:

[0023] (1) The above technical solution designed a shRNA targeting the IDE gene using the IDE gene as a template, packaged and constructed a recombinant lentiviral expression vector that specifically inhibits the expression of the IDE gene, and infected cells with the recombinant lentiviral expression vector that specifically inhibits the expression of the IDE gene, and successfully established an A549 cell line that knocks down the IDE gene, i.e., an A549 cell line that specifically inhibits the expression of the IDE gene. Compared with the control group, the IDE expression level in the A549 cell line that inhibits the expression of the IDE gene was significantly reduced. Therefore, it can be determined that the shRNA that specifically inhibits the expression of the IDE gene provided by the present invention has the function of specifically inhibiting the expression of the IDE gene.

[0024] (2) The above technical solution utilizes the H13N2 subtype influenza virus to infect the A549 cell line that specifically inhibits the expression of the IDE gene. By detecting the mRNA expression level of the NP gene of the H13N2 subtype influenza virus, it was found that the mRNA expression level of the NP gene in the A549 cell line that specifically inhibits the expression of the IDE gene was significantly lower than that in the control group. At the same time, by detecting the expression level of the PB2 protein of the H13N2 subtype influenza virus, it was found that the expression level of the PB2 protein in the A549 cell line that specifically inhibits the expression of the IDE gene was significantly lower than that in the control group. The viral titer after the H13N2 subtype influenza virus was infected with the A549 cell line that specifically inhibits the expression of the IDE gene was also significantly lower than that in the control group, thus proving that the specific inhibition of the expression of the IDE gene can significantly inhibit the replication of the H13N2 subtype influenza virus.

[0025] In summary, the above technical solution demonstrates for the first time that specifically inhibiting the expression of the IDE gene can significantly suppress the infection of H13N2 subtype influenza virus, providing an effective target for anti-influenza virus infection drugs and offering a new approach to antiviral infection, thus possessing good practical application value. Attached Figure Description

[0026] 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.

[0027] Figure 1 This is the sequencing result in Example 2 of the present invention.

[0028] Figure 2 This shows the mRNA expression of IDE after transfection of 293T cells with pLKO.1-CMV-copGFP-PURO-shIDE in Example 3 of this invention.

[0029] Figure 3 This shows the mRNA expression of IDE after it is packaged into lentivirus and then infects A549 in Example 3 of the present invention.

[0030] Figure 4 This shows the protein expression of IDE after shIDE-packaged lentivirus in Example 3 of the present invention infects A549.

[0031] Figure 5 This shows the mRNA expression of the NP gene of the H13N2 subtype influenza virus after IDE knockdown in Example 4 of the present invention.

[0032] Figure 6 This shows the protein expression of the PB2 gene of H13N2 subtype influenza virus after IDE knockdown in Example 4 of the present invention.

[0033] Figure 7 This shows the viral titer of the H13N2 subtype influenza virus after IDE knockdown in Example 4 of the present invention. Detailed Implementation

[0034] 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.

[0035] 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.

[0036] 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.

[0037] Example 1: Design and screening of shRNAs targeting IDE

[0038] (1) Design of shRNA targeting IDE

[0039] Using the IDE gene as a template (Gene ID: 5654), shRNAs for the target gene were designed using the online shRNA design website provided by Thermo Fisher Scientific. The sequences are shown in Table 1.

[0040] Table 1. Sequences corresponding to shRNA

[0041]

[0042] (2) shRNA primer annealing

[0043] 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.

[0044] Example 2: Construction of a recombinant lentiviral expression plasmid that specifically inhibits IDE gene expression

[0045] (1) pLKO.1-CMV-copGFP-PURO plasmid digestion

[0046] 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.

[0047] (2) pLKO.1-CMV-copGFP-PURO shRNA ligation and transformation

[0048] 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, this indicates a successful connection and conversion.

[0049] (3) Extraction of target plasmid

[0050] 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.

[0051] Example 3: Construction of an A549 cell line that specifically inhibits IDE gene expression

[0052] (1) shRNA knockdown plasmid was transfected into 293T cells to produce lentivirus.

[0053] HEK-293T cells were cultured, and when the cells reached approximately 80% confluency, the knockdown plasmid shRNA-IDE 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 h, 5 mL of culture medium was collected (stored at 4°C), and 5–6 mL of medium containing 10% serum-conjugated antibiotics was added.

[0054] 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.

[0055] RNA was extracted from the processed cell culture 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 the IDE. The results are as follows: Figure 2 As shown, the expression level of IDE mRNA in HEK-293T cells (pLKO.1-CMV-copGFP-PURO-shIDE-1) transfected with shRNA knockdown IDE plasmid was significantly lower than that in the control group (shNC), indicating that shIDE-1 has an inhibitory effect on IDE.

[0056] (2) Determination of the optimal concentration of puromycin

[0057] 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.

[0058] (3) Lentiviral infection of A549 cells

[0059] 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 every 24–48 hours, and 1.2 μg / mL of puromycin could be added for selection. A stable cell line would form in about one week.

[0060] (4) Identification of A549 cell lines that specifically inhibit IDE gene expression

[0061] 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 mRNA level of IDE. The results are as follows: Figure 3 As shown, the IDE mRNA expression level of the A549 cell line with IDE knockdown (pLKO.1-CMV-copGFP-PURO-shIDE-1) was significantly lower than that of the control group (shNC), indicating that the A549 cell line with IDE knockdown was successfully constructed.

[0062] Cells were lysed and the supernatant was collected, then subjected to SDS-PAGE electrophoresis, and subsequently transferred to a PVDF membrane for Western blot analysis to detect IDE protein expression. Results are as follows: Figure 4 As shown in the results, the expression level of IDE protein in the A549 cell line with IDE knockdown (shIDE) was significantly lower than that in the control group (shNC), indicating that the A549 cell line with IDE knockdown was successfully constructed.

[0063] Example 4: Effect of A549 cell line with specific inhibition of IDE gene expression on the proliferation of H13N2 subtype influenza virus

[0064] (1) A549 cell line that specifically inhibits IDE gene expression by H13N2 subtype influenza virus infection

[0065] When A549 cells (shNC) and A549 cell lines specifically inhibiting IDE gene expression (shIDE) reached approximately 80% confluence, H13N2 subtype influenza virus (0.5 MOI) was inoculated into the cells. After incubation in an incubator for 1 hour, F12K medium containing TPCK was added to achieve a final TPCK concentration of 2 μg / mL. After 24 hours of culture, viral replication was assessed.

[0066] (2) Virus replication detection

[0067] (2.1) Quantitative PCR detection of viral NP gene expression level

[0068] 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 5 As shown, the results indicate that the mRNA level of the NP gene in A549 cells infected with H13N2 subtype influenza virus that specifically inhibits IDE gene expression was significantly lower than that in the A549 cell control group (shNC). This result suggests that inhibiting IDE expression can significantly suppress the replication of H13N2 subtype influenza virus.

[0069] (2.2) Western blot detection of viral PB2 protein expression level

[0070] 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 6 As shown, the results indicated that the amount of PB2 protein in A549 cells infected with H13N2 subtype influenza virus that specifically inhibited IDE gene expression was significantly lower than that in the A549 cell control group (shNC). This result suggests that inhibiting IDE expression can significantly suppress the replication of H13N2 subtype influenza virus.

[0071] (2.3) Detection of viral titers of H13N2 subtype influenza virus in different cell lines

[0072] Supernatants were collected 24 h after H13N2 subtype influenza virus infection of A549 cells (shNC) and A549 cell lines specifically inhibiting IDE gene expression (shIDE). The collected supernatants were serially diluted 10-fold (dilution factor 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 7As shown, the results indicated that the viral titer of H13N2 subtype influenza virus in the A549 cell line (shIDE) with specifically suppressed IDE gene expression was significantly lower than that in the A549 cell group (shNC). This result suggests that inhibiting IDE expression can reduce the viral titer of H13N2 subtype influenza virus.

[0073] 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. Use of a biomaterial that inhibits the expression of an IDE gene in the manufacture of an anti-influenza virus product; wherein, The biological material for inhibiting the expression of the IDE gene is an shRNA, a recombinant lentivirus expression plasmid containing the shRNA, or a host cell containing the shRNA. The sense strand of the shRNA is shown in SEQ ID NO. 1, and the antisense strand of the shRNA is shown in SEQ ID NO.

2. The influenza virus is an H13N2 subtype influenza virus.

2. Use according to claim 1, wherein The recombinant lentivirus expression plasmid is obtained by cloning the shRNA into a lentivirus plasmid vector.

3. The use according to claim 1, wherein the compound is ###0002### The host cell is a cell infected by a recombinant lentivirus to specifically inhibit the expression of the IDE gene; the recombinant lentivirus is obtained by packaging after transfecting 293T cells with a recombinant lentivirus expression vector; the recombinant lentivirus expression vector contains the recombinant lentivirus expression plasmid and a helper plasmid; the helper plasmid includes psPAX2 and pMD2.G.

4. The use according to claim 1, characterized in that, The anti-influenza virus product is a drug or a general test reagent for non-medical use. The anti-influenza virus product specifically inhibits the proliferation and replication of an H13N2 subtype influenza virus.

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