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

Abstract

The invention belongs to the technical field of gene engineering and biological medicine, and particularly relates to a biological material for inhibiting IDE gene expression and application of the biological material in anti-influenza virus. Researches find that specific inhibition of expression of the IDE gene can significantly inhibit infection of influenza viruses, especially H13N2 subtype influenza viruses. Specifically, the research finds that the mRNA expression level of the NP gene of the H13N2 subtype influenza virus in the A549 cell line for specifically inhibiting the IDE gene expression and the expression level of the PB2 protein are obviously reduced; meanwhile, the virus titer after the H13N2 subtype influenza virus infects the A549 cell line specifically inhibiting IDE gene expression is also obviously lower than that of a control group. The invention proves that the infection of the H13N2 subtype influenza virus can be obviously inhibited by specifically inhibiting the expression of the IDE gene, and a new thought is provided for resisting influenza virus infection, so that the method has a good practical application value.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering and biomedicine technology, and particularly relates to a biomaterial for inhibiting IDE gene expression and its application in resisting influenza virus. Background Art

[0002] Influenza viruses are segmented RNA viruses that can be divided into 18 HA subtypes and 11 NA subtypes based on differences in the hemagglutinin (HA) and neuraminidase (NA) on their surface. Influenza viruses can also be categorized into human and avian influenza viruses, depending on the host they infect. Host factors play a crucial role in influenza virus infection and replication, and therefore, studying the interactions between host factors and influenza viruses is crucial for the prevention and control of the virus.

[0003] Insulin-degrading enzyme (IDE) is an enzyme widely expressed in various human tissues, playing a particularly crucial role in the brain. Its primary function is to degrade insulin and Aβ protein, maintaining a balance between insulin and Aβ, protecting nerve cells from damage, and thus maintaining 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 exacerbates Aβ deposition in the brain, triggering synaptic damage and neuronal dysfunction, ultimately leading to cognitive decline. However, the inventors have discovered no reports of IDE being used against influenza viruses. Summary of the Invention

[0004] To address the shortcomings of the prior art, the present invention aims to provide biomaterials that inhibit IDE gene expression and their use in combating influenza viruses. Specifically, the present invention has discovered that specifically inhibiting IDE gene expression can significantly inhibit influenza virus infection, particularly infection by the H13N2 subtype. Based on these 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: In a first aspect, the present invention provides a biomaterial for inhibiting IDE gene expression. The biomaterial may be a short hairpin RNA (shRNA), a recombinant lentiviral expression plasmid, a recombinant lentiviral expression vector or a host cell.

[0006] The shRNA includes a sense chain and an antisense chain, wherein the sense chain has a nucleotide sequence as shown in SEQ ID NO.1; the corresponding antisense chain has a nucleotide sequence as shown in SEQ ID NO.2; or, the sense chain has a nucleotide sequence as shown in SEQ ID NO.3, and the corresponding antisense chain has a nucleotide sequence as shown in SEQ ID NO.4.

[0007] 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.

[0008] 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.

[0009] 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.

[0010] The host cell may be a cell infected with a recombinant lentivirus to specifically inhibit IDE gene expression. In one embodiment of the present invention, the host cell may be an A549 cell. The recombinant lentivirus is obtained by transfecting 293T cells with the recombinant lentiviral expression vector described above and packaging the resultant cells.

[0011] The present invention has been demonstrated through experiments that, compared with the control group, the host cells can significantly inhibit the replication of the H13N2 subtype influenza virus and reduce the titer of the H13N2 subtype influenza virus after being infected by the H13N2 subtype influenza virus, and therefore can be used as an anti-influenza virus product.

[0012] Therefore, the second aspect of the present invention provides the use of the above-mentioned biomaterial for inhibiting IDE gene expression in the preparation of anti-influenza virus products.

[0013] In the present invention, the influenza virus is further an H13N2 subtype influenza virus.

[0014] The anti-influenza virus product can be a medicine or a common test reagent for non-medical use, and the common test reagent can be used for basic research on the relationship between the IDE gene and influenza virus.

[0015] Specifically, the anti-influenza virus product specifically inhibits the proliferation and replication of the H13N2 subtype influenza virus, thereby significantly inhibiting the infection of the H13N2 subtype influenza virus.

[0016] When the anti-influenza virus product is a drug, the drug can be a drug for preventing influenza virus infection; further, the drug further comprises a pharmaceutically acceptable excipient and / or carrier, and the content of the excipient and / or carrier 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, which are not specifically limited herein.

[0017] The dosage form of the Chinese medicine preparation of the present invention is prepared by conventional preparation methods into a conventional liquid dosage form or solid dosage form acceptable for pharmaceutical use. Specifically, the dosage form of the medicine can be oral tablets, oral liquids, syrups, granules, capsules, tablets, or pills. As a result, the medicine is easy to carry and take, which is conducive to clinical promotion and practical application.

[0018] In order to realize the above dosage forms, pharmaceutically acceptable excipients need to be added when preparing these dosage forms, for example: ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycerol, sorbitol, potassium sorbate, partial glyceride mixtures 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, polyvinyl pyrrolidone, etc.

[0019] Tablets and capsules for oral administration may contain excipients such as binders, such as syrup, gum arabic, sorbitol, tragacanth, or polyvinyl pyrrolidone, 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. Tablets for oral administration may be coated by methods well known in the pharmaceutical industry.

[0020] Oral liquids can be prepared as suspensions, solutions, emulsions, syrups, etc. of water and oil, or as dry products that are supplemented with water or other suitable media before use. Such liquid preparations can contain conventional additives such as suspending agents, sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel, hydrogenated edible oils and fats, emulsifiers such as lecithin, sorbitan monooleate, and gum arabic; or non-aqueous vehicles including almond oil, oils such as glycerol, ethylene glycol, or ethanol; and preservatives including methyl parahydroxybenzoate, propyl parahydroxybenzoate, or sorbic acid.

[0021] Beneficial technical effects of one or more of the above technical solutions: (1) The above technical solution uses the IDE gene as a template to design shRNA targeting the IDE gene, packages and constructs a recombinant lentiviral expression vector that specifically inhibits the expression of the IDE gene, and infects cells with the recombinant lentiviral expression vector that specifically inhibits the expression of the IDE gene. A549 cell line that knocks down the IDE gene, i.e., an A549 cell line that specifically inhibits the expression of the IDE gene, is successfully established. Compared with the control group, the IDE expression level in the A549 cell line that inhibits the expression of IDE is 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.

[0022] (2) The above technical scheme uses 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 is found that the expression level of the NP gene mRNA in the A549 cell line that specifically inhibits the expression of the IDE gene is 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 is found that the expression level of the PB2 protein in the A549 cell line that specifically inhibits the expression of the IDE gene is significantly lower than that in the control group; the virus titer of the A549 cell line that specifically inhibits the expression of the IDE gene after infection with the H13N2 subtype influenza virus is also significantly lower than that in the control group, further proving that specifically inhibiting the expression of the IDE gene can significantly inhibit the replication of the H13N2 subtype influenza virus.

[0023] In summary, the above technical solution has demonstrated for the first time that specifically inhibiting the expression of the IDE gene can significantly inhibit the infection of the H13N2 subtype influenza virus, providing an effective target for anti-influenza virus infection drugs and a new idea for anti-viral infection, so it has good 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 2 This is the mRNA expression of IDE after 293T cells were transfected with pLKO.1-CMV-copGFP-PURO-shIDE in Example 3 of the present invention.

[0027] Figure 3 This is the mRNA expression of IDE after the shIDE in Example 3 of the present invention was packaged into lentivirus and infected A549 cells.

[0028] Figure 4 This is the protein expression of IDE after the shIDE in Example 3 of the present invention was packaged into lentivirus and infected A549 cells.

[0029] Figure 5 This is the mRNA expression of the H13N2 subtype influenza virus NP gene after knocking down IDE in Example 4 of the present invention.

[0030] Figure 6 This is the protein expression of the PB2 gene of the H13N2 subtype influenza virus after knocking down IDE in Example 4 of the present invention.

[0031] Figure 7 This is the viral titer of the H13N2 subtype influenza virus after knocking down IDE in Example 4 of the present invention. DETAILED DESCRIPTION

[0032] 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.

[0033] 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.

[0034] 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.

[0035] Example 1 Design and screening of shRNA targeting IDE (1) Design of shRNA targeting IDE Using the IDE gene as a template (Gene ID: 5654), shRNA targeting the target gene was designed using the online shRNA design website provided by Thermo Fisher Scientific. The sequences are shown in Table 1.

[0036] Table 1 Sequences corresponding to shRNA

[0037] (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.

[0038] Example 2 Construction of a recombinant lentiviral expression plasmid that specifically inhibits IDE 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.

[0039] (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 As shown, it indicates successful connection and transformation.

[0040] (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.

[0041] Example 3 Construction of A549 cell line specifically inhibiting IDE 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 knockdown plasmid shRNA-IDE and the helper plasmids psPAX2 and pMD2.G at a concentration 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 antibodies.

[0042] 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.

[0043] The treated cell fluid was used to extract cellular RNA 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 determine the mRNA level of IDE. The results are shown in Figure 2. Figure 2 As shown in the figure, the IDE mRNA expression level in HEK-293T cells transfected with shRNA knockdown plasmid (pLKO.1-CMV-copGFP-PURO-shIDE-1) was significantly lower than that in the control group (shNC), indicating that shIDE-1 has an inhibitory effect on IDE.

[0044] (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.

[0045] (3) Lentivirus infection of A549 cells A549 cells were plated in 6-well plates. When cells reached approximately 80% confluence, the packaged lentivirus was added (MOI = 5). 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.

[0046] (4) Identification of A549 cell lines that specifically inhibit IDE 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 IDE. The results are shown in Figure 2. Figure 3 As shown in the figure, the IDE mRNA expression level of the IDE knockdown A549 cell line (pLKO.1-CMV-copGFP-PURO-shIDE-1) was significantly lower than that of the control group (shNC), indicating that the IDE knockdown A549 cell line was successfully constructed.

[0047] The cells were lysed and the supernatant was collected and subjected to SDS-PAGE electrophoresis. The supernatant was then transferred to a PVDF membrane and Western blot was performed to detect the protein expression of IDE. Figure 4 As shown, the results showed that the IDE protein expression level of the A549 cell line with IDE knockdown (shIDE) was significantly lower than that of the control group (shNC), indicating that the A549 cell line with IDE knockdown was successfully constructed.

[0048] Example 4 Effect of A549 Cell Line with Specific Inhibition of IDE Gene Expression on the Proliferation of H13N2 Subtype Influenza Virus (1) Infection of H13N2 subtype influenza virus in A549 cell line specifically inhibiting IDE gene expression A549 cells (shNC) and an A549 cell line specifically inhibiting IDE gene expression (shIDE) were cultured to approximately 80% confluence and inoculated with H13N2 subtype influenza virus (MOI) at 0.5. After incubation for 1 hour in an incubator, 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 5As shown, the results showed that the mRNA level of NP gene in A549 cells infected with H13N2 subtype influenza virus and specifically inhibiting IDE gene expression was significantly lower than that in the A549 cell control group (shNC). This result indicates that inhibiting IDE expression can significantly inhibit the replication of H13N2 subtype influenza virus.

[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 6 The results showed that the amount of PB2 protein in A549 cells infected with H13N2 subtype influenza virus and specifically inhibiting IDE gene expression was significantly lower than that in the A549 cell control group (shNC). This result indicates that inhibiting IDE expression can significantly inhibit the replication of H13N2 subtype influenza virus.

[0051] (2.3) Detection of viral titers of H13N2 subtype influenza virus in different cell lines The supernatants of A549 cells (shNC) and A549 cell line with specific inhibition of IDE gene expression (shIDE) were collected 24 hours after infection with H13N2 subtype influenza virus. The collected supernatants were serially 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 7 The results showed that the viral titer of H13N2 subtype influenza virus in the A549 cell line with specific inhibition of IDE gene expression (shIDE) was significantly lower than that in the A549 cell group (shNC). This result indicates that inhibiting IDE expression can reduce the viral titer of H13N2 subtype influenza virus.

[0052] 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 inhibiting IDE gene expression, characterized in that: The biological material is shRNA, recombinant lentiviral expression plasmid, recombinant lentiviral expression vector or host cell; The shRNA includes a sense chain and an antisense chain, wherein the sense chain has a nucleotide sequence as shown in SEQ ID NO.1; the corresponding antisense chain has a nucleotide sequence as shown in SEQ ID NO.2; or, the sense chain has a nucleotide sequence as shown in SEQ ID NO.3, and the corresponding antisense chain has a nucleotide sequence as shown in SEQ ID NO.

4.

2. The biomaterial according to claim 1, wherein 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.

3. The biomaterial according to claim 1, wherein The recombinant lentiviral expression plasmid is obtained by cloning the shRNA into a lentiviral plasmid vector.

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 cell is a cell infected by a recombinant lentivirus, thereby specifically inhibiting the expression of the IDE gene.

6. The biomaterial according to claim 5, wherein The host cell is an A549 cell; the recombinant lentivirus is obtained by transfecting a recombinant lentiviral expression vector into 293T cells and then packaging the vector.

7. Use of the biomaterial for inhibiting IDE gene expression according to any one of claims 1 to 6 in the preparation of an anti-influenza virus product.

8. The use according to claim 7, characterized in that The influenza virus is an H13N2 subtype influenza virus.

9. The use according to claim 7, characterized in that The anti-influenza virus product is a drug or a common test reagent for non-medical purposes; The anti-influenza virus product specifically inhibits the proliferation and replication of the H13N2 subtype influenza virus.

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