Application of TNFAIP1 as target protein in preparation of anti-influenza virus drugs
The preparation of pharmaceutically acceptable dosage forms by overexpressing the TNFAIP1 gene has been solved, and the problem of difficult inhibition of influenza A virus replication in the prior art has been solved, and the effect of significantly inhibiting influenza viruses in different cells is achieved, and it is applied to the preparation of anti-influenza virus drugs and vaccine adjuvants.
Patent Information
- Application Number
- CN202510252879.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-05
- Publication Date
- 2025-07-04
AI Technical Summary
The prior art is difficult to effectively inhibit the replication of influenza A virus, especially in the cross-species transmission of different subtypes of influenza viruses, which affects human health and the development of livestock and poultry industry.
By overexpressing the host TNFAIP1 gene and using TNFAIP1 as the target protein, pharmaceutically acceptable dosage forms such as powder injections, capsules, tablets, suspensions, etc. are prepared for the preparation of anti-influenza virus drugs; TNFAIP1 or its pharmaceutically acceptable salts are used as adjuvant in influenza virus vaccines, and oil emulsion adjuvant or aluminum adjuvant is used.
Adding TNFAIP1 to cultured cells can significantly reduce the replication level of influenza A virus. The prepared drugs or adjuvant can effectively inhibit the replication of influenza virus and are used to prepare drugs or adjuvants that are anti-IAV infection.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology, and particularly relates to the application of TNFAIP1 as a target protein in the preparation of anti-influenza virus drugs. Background Art
[0002] Influenza A virus (IAV) is an important zoonotic pathogen that causes global influenza pandemics and seasonal influenza outbreaks. Due to the high infectivity of influenza virus, it is extremely prone to epidemics, even worldwide pandemics. In recent years, the emergence of highly pathogenic H5 and H7 subtypes and the cross-species transmission of different subtypes of influenza virus have continuously threatened human life and health and affected the development of the livestock and poultry industry. Therefore, analyzing the pathogenic mechanism of influenza virus and finding new antiviral targets are crucial for the prevention and control of influenza virus.
[0003] Tumor necrosis factor α-induced protein 1 (TNFAIP1) is involved in many important processes such as DNA synthesis, damage and repair, induction of differentiation and apoptosis, and inflammation. Existing studies have reported that TNFAIP1 can function as a substrate-specific adaptor protein to exert the function of ubiquitin ligase. TNFAIP1 is involved in a variety of life activities and signal transduction, such as the innate immune pathway, the inflammatory pathway, and apoptosis of cells.
[0004] The present invention discovers that by overexpressing the host TNFAIP1 gene, the replication of different subtypes of influenza virus can be inhibited; overexpressing the host TNFAIP1 gene inhibits the replication of influenza virus in different cells; knocking down TNFAIP1 significantly promotes the replication of influenza virus. It indicates that TNFAIP1 can be used as a target for the preparation of drugs or adjuvants that inhibit influenza virus. Summary of the Invention
[0005] The purpose of the present invention is to provide the application of TNFAIP1 as a target protein in the preparation of anti-influenza virus drugs.
[0006] Another purpose of the present invention is to provide the application of TNFAIP1 protein or its pharmaceutically acceptable salt in the preparation of influenza virus vaccine adjuvants.
[0007] The technical solution of the present invention is as follows: (1) The application of TNFAIP1 as a target protein in the preparation of anti-influenza virus drugs.
[0008] Further, the influenza virus is Influenza A virus.
[0009] Further, TNFAIP1 or its pharmaceutically acceptable salt is added to a pharmaceutically acceptable carrier and / or excipient to form any pharmaceutically acceptable dosage form.
[0010] Further, the dosage forms include powder injections, capsules, tablets, and suspensions.
[0011] (2) Use of TNFAIP1 protein or a pharmaceutically acceptable salt thereof in the preparation of an adjuvant for an influenza virus vaccine.
[0012] Further, the influenza virus is an influenza A virus.
[0013] Further, TNFAIP1 or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier and / or excipient to form any pharmaceutically acceptable dosage form.
[0014] Further, the dosage form includes an oil-in-water emulsion adjuvant and an aluminum adjuvant.
[0015] The beneficial effect of the present invention is that adding TNFAIP1 to the cells for culturing IAV can reduce the replication level of IAV, indicating that TNFAIP1 has the effect of inhibiting the replication of IAV and can be used for the preparation of drugs or adjuvants for anti-IAV infection to inhibit the replication of IAV. Description of the Drawings
[0016] Figure 1 It is a result diagram for detecting the effect of overexpressing TNFAIP1 protein on different doses of influenza A virus H1N1 in human lung cancer alveolar epithelial cells A549; Figure 2 It is a result diagram for detecting the effect of overexpressing TNFAIP1 protein on the infection of human lung cancer alveolar epithelial cells A549 by influenza virus subtypes H3N2, H6N6 and H9N2; Figure 3 It is a result diagram for detecting the effect of knocking down TNFAIP1 protein on the infection of human lung cancer alveolar epithelial cells A549 by H1N1 influenza virus; Figure 4 It is a result diagram for detecting the effect of overexpressing TNFAIP1 protein on the replication of H1N1 subtype influenza virus in hamster kidney cells BHK-21, porcine kidney cells PK-15 and human osteosarcoma cells U2OS; Figure 5 It is a diagram of the interaction between TNFAIP1 and influenza A virus proteins; Figure 6 It is a result diagram for determining the effect of TNFAIP1 on the polymerase activity of influenza virus by a dual-luciferase reporter gene activity experiment; Figure 7 It is a result diagram for determining the effect of overexpressing TNFAIP1 on the stability of PB1 by an immunoblotting experiment. Specific Embodiments
[0017] The present invention will be further described in detail below with reference to the drawings and specific embodiments.
[0018] Example 1: Effect of overexpressing TNFAIP1 on the replication of influenza A virus at different doses.
[0019] 1. Cell transfection and virus infection A strain of human lung cancer alveolar epithelial cells A549 was seeded into a 12-well plate containing DMEM medium (containing 9% fetal bovine serum and 1% penicillin / streptomycin), and cultured in a carbon dioxide incubator (37 °C, 5% CO2) until the cell density reached approximately 1×10 6 cell / mL. Then, the Flag-TNFAIP1 expression plasmid was transfected, and the cells were co-cultured for another 24 h. The A / H1N1 strain of influenza A virus (MOI = 0.01 / MOI = 3) was infected at 500 μL / well. After 1 h, the virus was discarded, and the cells were washed 3 times with PBS and then cultured in the presence of 0.05 μg / mL of TPCK trypsin. The cell supernatants were collected at 12, 24, 48, 72 h or 6, 12, 24, 24 h.
[0020] 2. Virus titer detection (PFU) The PFU method was used to evaluate the replication efficiency of the H1N1 subtype influenza virus, and the experiment was set with 3 replicates. After the cell supernatants were serially diluted 10-fold 10 times, 100 μL of the samples at the dilution factors of 10 -3 、10 -4 、10 -5 were added to MDCK cells. After 1 h, the virus solution was discarded, and the cells were washed once with PBS and then cultured in agar-DMEM medium containing 1 μg / mL of TPCK. After solidification, the plates were inverted and cultured. After 72 h, the number of plaques was recorded.
[0021] 3. Results Figure 1 To detect the effect of overexpressing TNFAIP1 protein on the replication of influenza A virus H1N1 at different doses in human lung cancer alveolar epithelial cells A549, Figure A shows the growth of the virus-infected cells when the virus infection dose is MOI = 0.01, and Figure B shows the growth of the virus-infected cells when the virus infection dose is MOI = 3. Among them, "h. p. i." is the time after virus infection (in hours); to detect the replication of the virus-infected cells, "EV" is the empty vector control (human lung cancer alveolar epithelial cells A549 transfected with the empty plasmid), and "Flag-TNFAIP1" is human lung cancer alveolar epithelial cells A549 transfected with the plasmid vector overexpressing TNFAIP1 protein. It can be Figure 1 seen that by making the virus growth curve, overexpressing TNFAIP1 can significantly inhibit virus replication at different time points after infection with influenza virus at different doses compared with the control group.
[0022] Example 2: Effect of overexpressing TNFAIP1 on the replication of influenza virus of different subtypes.
[0023] 1. Cell transfection and virus infection Cells were cultured and transfected according to the method in Example 1. Cells were infected with influenza A virus strains H3N2, H6N6, and H9N2 (MOI = 0.01) at 500 μL / well. After 1 h, the virus was discarded, and the cells were washed 3 times with PBS and then cultured with TPCK trypsin containing 0.05 μg / mL. The cell supernatant was collected at 48 h.
[0024] 2. Virus titer detection (PFU) The PFU method was used to evaluate the replication efficiency of influenza virus subtypes H3N2, H6N6, and H9N2. The experiment was set up with 3 replicates. After 10-fold serial dilution of the cell supernatant, 100 μL of the samples at dilutions of 10 -3 、10 -4 、10 -5 were added to MDCK cells. After 1 h, the virus solution was discarded, and the cells were washed once with PBS and then cultured with agar-DMEM medium containing 1 μg / mL TPCK. After solidification, the plates were incubated upside down. The number of plaques was recorded after 72 h.
[0025] 3. Results Figure 2 The figure shows the results of detecting the effect of overexpressing TNFAIP1 protein on the infection of human lung cancer alveolar epithelial cells A549 by different subtypes of influenza virus H3N2, H6N6, and H9N2. Among them, "h. p. i." is the time after virus infection (in hours); to detect the replication of the virus-infected cells, "EV" is the empty vector control (human lung cancer alveolar epithelial cells A549 transfected with the empty plasmid), and "Flag-TNFAIP1" is the human lung cancer alveolar epithelial cells A549 transfected with the plasmid vector overexpressing TNFAIP1 protein. As Figure 2 can be seen, by making the virus growth curve, overexpression of TNFAIP1 can significantly inhibit virus replication compared with the control group after infection with different subtypes of influenza virus.
[0026] Example 3: Effect of knocking down TNFAIP1 on influenza virus replication.
[0027] 1. Cell transfection and virus infection The A549-sh-TNFAIP1 cell line was cultured according to the method in Example 1. When the cell density reached 1×10 6 cell / mL, the cells were infected with H1N1 strain (MOI = 0.01) at 500 μL / well. After 1 h, the virus was discarded, and the cells were washed 3 times with PBS and then cultured with TPCK trypsin containing 0.05 μg / mL. The cell supernatant was collected at 12, 24, 48, and 72 h.
[0028] 2. Virus Titer Detection (PFU) The PFU method was used to evaluate the replication efficiency of H1N1 subtype influenza virus, and 3 replicates were set in the experiment. After 10-fold serial dilution of the cell supernatant, 100 μL of the samples at dilutions of 10 -3 、10 -4 、10 -5 were added to MDCK cells. After 1 h, the virus solution was discarded, and after washing once with PBS, agar-DMEM medium containing 1 μg / mL TPCK was added. After solidification, the culture was inverted, and the number of plaques was recorded after 72 h.
[0029] 3. Results Figure 3 The figure shows the results of detecting the effect of knocking down TNFAIP1 protein on the infection of human lung cancer alveolar epithelial cells A549 by H1N1 influenza virus. Among them, "h. p. i." is the time after virus infection (unit: hour); to detect the replication of the virus-infected cells, "EV" is the empty vector control (human lung cancer alveolar epithelial cells A549 transfected with the empty plasmid), and "Flag-TNFAIP1" is human lung cancer alveolar epithelial cells A549 transfected with the plasmid vector overexpressing TNFAIP1 protein. As Figure 3 can be seen, by making the virus growth curve, knocking down TNFAIP1 can significantly promote virus replication compared with the control group after H1N1 influenza virus infection.
[0030] Example 4: Effects of overexpressing TNFAIP1 on influenza virus replication in different cells.
[0031] 1. Cell Transfection and Virus Infection Hamster kidney fibroblast cells BHK-21, porcine kidney cells PK-15, and human osteosarcoma cells U2OS were seeded in a 12-well plate containing DMEM medium (containing 9% fetal bovine serum and 1% penicillin / streptomycin), and cultured in a carbon dioxide incubator (37 °C, 5% CO2) until the cell density reached about 1×10 6 cells / mL. Flag-TNFAIP1 expression plasmid was transfected, and co-cultured with the cells for another 24 h. 500 μL / well of influenza A virus H1N1 (MOI = 0.01) was infected. After 1 h, the virus was discarded, and after washing 3 times with PBS, culture with 0.05 μg / mL of TPCK trypsin was added, and the cell supernatant was collected at 48 h.
[0032] 2. Virus Titer Detection (PFU) The PFU method was used to evaluate the replication efficiency of H1N1 subtype influenza virus, and 3 replicates were set in the experiment. After 10-fold serial dilution of the cell supernatant, 10 -3 、10 -4 、10-5 100 μL of each dilution of the sample was added to MDCK cells. After 1 h, the virus solution was discarded. After washing once with PBS, agar-DMEM medium containing 1 μg / mL TPCK was added. After solidification, the culture was inverted and cultured. After 72 h, the number of plaques was recorded.
[0033] 3. Results Figure 4 It is a structural diagram for detecting the effect of overexpressing TNFAIP1 protein on the replication of H1N1 subtype influenza virus in hamster kidney cells BHK-21, porcine kidney cells PK-15 and human osteosarcoma cells U2OS. Among them, "h. p. i." is the time after virus infection (unit: hour); to detect the replication of the virus-infected cells, "EV" is the empty vector control (hamster kidney cells BHK-21, porcine kidney cells PK-15 and human osteosarcoma cells U2OS transfected with the empty plasmid), and "Flag-TNFAIP1" is the hamster kidney cells BHK-21, porcine kidney cells PK-15 and human osteosarcoma cells U2OS transfected with the plasmid vector overexpressing TNFAIP1 protein. From Figure 4 It can be seen that by making the virus growth curve, overexpression of TNFAIP1 inhibits the replication of H1N1 subtype influenza virus in BHK-21, PK-15 and U2OS cells.
[0034] Example 5: Determination of the interaction between TNFAIP1 and influenza virus proteins.
[0035] In the present invention, the interaction between TNFAIP1 and influenza virus was determined by immunoprecipitation experiment screening.
[0036] 1. Cultivate 293T cells according to the method in Example 1 until the density is about 1×10 6 cell / mL, then transfect the HA-TNFAIP1 expression plasmid into the cells. After culturing for 24 h, infect with PR8 (MOI = 5), and collect the cells after 24 h. After washing once with PBS, add NP40 lysis buffer to the cells to fully lyse them, sonicate at 80 W for 1 min at 4 °C, and centrifuge at 12000 rpm. Divide the supernatant into two equal parts. Take 300 μL of one part and add protein A / G conjugated HA antibody or IgG antibody respectively, and incubate overnight at 4 °C. Wash 3 times with NP40 + 5M NaCl and then denature at 100 °C for 10 min with 2×loading buffer; take 100 μL of the other part as the whole cell lysate (WCL) and denature at 100 °C for 10 min with 2×loading buffer, and perform immunoblotting analysis respectively.
[0037] 2. Results Figure 5Figure showing the interaction between TNFAIP1 and influenza A virus proteins, which shows the influenza virus proteins that interact with TNFAIP1 after PR8 infection determined by co-immunoprecipitation experiments. A shows human kidney epithelial cells 293T transfected with the plasmid vector for overexpression of TNFAIP1 protein "HA-TNFAIP1". Among them, "IB: PB1 / PB2 / PA / NP / HA / NS1 / NS2 / M1 / M2" is to detect the expression of each viral protein, "IB: HA" is to detect the HA tag, "IP: HA" is to capture the interacting proteins by magnetic beads labeled with HA-tagged antibody, and "WCL" is the total protein in the cell lysate supernatant; B shows the interaction between TNFAIP1 and PB1 at different time points after PR8 infection. Among them, "h. p, i" is the time after virus infection (in hours).
[0038] As can be seen from Figure 5 A, after influenza virus infection, TNFAIP1 only interacts with the polymerase protein PB1.
[0039] 3. The present invention further determines the interaction between TNFAIP1 and PB1 through an endogenous co-immunoprecipitation experiment. 293T cells were cultured according to the method in Example 1 until the density was about 1×10 6 cell / mL, and then the cells were infected with H1N1 subtype influenza virus (MOI = 5). The cells were collected at different time points 0, 6, 12, and 24 h. After washing once with PBS, NP40 lysis buffer was added to the cells to fully lyse them. They were sonicated at 80 W for 1 min at 4 °C and centrifuged at 12,000 rpm. The supernatant was divided into two parts. One part, 300 μL was taken and added to protein A / G conjugated with TNFAIP1 antibody or IgG antibody, and incubated overnight at 4 °C. After washing 3 times with NP40 + 5M NaCl, it was denatured at 100 °C for 10 min with 2×loading; one part, 100 μL was taken as the whole cell lysate (WCL) and denatured at 100 °C for 10 min with 2×loading, and immunoblotting experiments were respectively carried out for analysis.
[0040] 4. Figure 5 The results of B show that at different time points after virus infection, TNFAIP1 interacts with PB1, but does not interact with the control protein NP, indicating that TNFAIP1 specifically interacts with the polymerase protein PB1.
[0041] Example 6: Effects of overexpression and knockdown of TNFAIP1 on the polymerase activity of influenza A virus.
[0042] 1. Figure 6Determine the result graph of the effect of TNFAIP1 on the polymerase activity of influenza virus for the dual-luciferase reporter gene activity experiment. Among them, A shows the effect of overexpressing TNFAIP1 on the polymerase activity of influenza virus, and "+" indicates transfection of the corresponding plasmid vector; B shows the effect of knocking down TNFAIP1 on the polymerase activity of influenza virus, "+" indicates transfection of the corresponding plasmid vector, and "si-TNFAIP1" represents cells transfected with TNFAIP1 gene-specific siRNA.
[0043] Figure 6 In A, 293T cells were cultured according to the method in Example 1 until the density reached about 1×10 6 cell / mL, and then the Flag-TNFAIP1 expression plasmid, the polymerase protein pCAGGS-PB1 / PB2\PA\NP expression plasmid, and the luciferin-labeled NP-Luc expression plasmid were co-transfected into the cells. After culturing for 24 h, cell protein samples were collected. According to the instructions of the dual-luciferase reporter kit (Promega, WI, USA), cell lysates were prepared using passive lysis buffer, and the firefly fluorescence and Renilla luciferase activities were detected. When analyzing the data, the firefly luciferase activity was normalized according to the Renilla luciferase activity, and the relative reporter gene activity was calculated with the value of the negative control well as 1. All experiments were repeated three times.
[0044] Figure 6 In B, 293T cells were cultured according to the method in Example 1 until the density reached about 1×10 6 cell / mL, and then the TNFAIP1 gene-specific siRNA, the polymerase protein pCAGGS-PB1 / PB2\PA\NP expression plasmid, and the luciferin-labeled NP-Luc expression plasmid were co-transfected into the cells. After culturing for 24 h, cell protein samples were collected. According to the instructions of the dual-luciferase reporter kit (Promega, WI, USA), cell lysates were prepared using passive lysis buffer, and the firefly fluorescence and Renilla luciferase activities were detected. When analyzing the data, the firefly luciferase activity was normalized according to the Renilla luciferase activity, and the relative reporter gene activity was calculated with the value of the negative control well as 1. All experiments were repeated three times.
[0045] The siRNA used was synthesized by Guangzhou Ribobio Co., Ltd., and the nucleotide sequence of the TNFAIP1 gene-specific siRNA is shown in SEQ ID NO.1.
[0046] SEQ ID NO.1: GCAACAACAAGTATTCCTA.
[0047] 2. Results The results showed thatFigure 6 Overexpression of TNFAIP1 in A can significantly down-regulate the polymerase activity of influenza virus in a dose-dependent manner. Figure 6 B further confirmed that after knocking down TNFAIP1, the polymerase activity of influenza virus was restored. This indicates that TNFAIP1 may inhibit virus replication by affecting polymerase activity.
[0048] Example 7: Effect of overexpressing TNFAIP1 on the stability of PB1.
[0049] 1. Cultivate 293T cells according to the method in Example 1 until the density is about 1×10 6 cell / mL, then transfect the cells with Flag-TNFAIP1 expression plasmid, Myc-NP expression plasmid, Myc-PB1 expression plasmid and HA-β-actin expression plasmid. Collect the cells 24 h after transfection. Wash the cells once with PBS, then add RIPA lysis buffer to fully lyse the cells, sonicate at 80 W for 1 min at 4 °C, centrifuge at 12,000 rpm, add 2×SDS-loading buffer for denaturation, and then perform immunoblotting analysis.
[0050] 2. Results Figure 7 This is the result diagram of the immunoblotting experiment to determine the effect of overexpressing TNFAIP1 on the stability of PB1. Among them, "FLAG-TNFAIP1" is the plasmid vector transfected to express TNFAIP1 protein with a Flag tag. From left to right, the amount of the above-mentioned expression plasmid transfected gradually increases (0 ng, 200 ng, 400 ng, 800 ng); "Myc-PB1" is the plasmid vector transfected to overexpress PB1 protein; "Myc-NP" is the plasmid vector transfected to overexpress NP protein; "HA-β-actin" is the plasmid vector transfected to overexpress the internal reference gene β-actin protein; "IB: Flag" is to detect the expression of the protein TNFAIP1 with the tag Flag; "IB: Myc" is to detect the expression of the proteins PB1 and NP with the tag Myc; "IB: HA" is to detect the transfection efficiency of the internal reference protein β-actin with the tag HA. Figure 7 The results show that as the dose of TNFAIP1 increases, the expression level of PB1 decreases, and the expression of HA-β-actin is consistent, indicating that TNFAIP1 down-regulates the stability of PB1 under the same transfection efficiency. TNFAIP1 may directly degrade PB1, resulting in a decrease in the polymerase activity of the virus and inhibiting virus replication.
Claims
1. Use of TNFAIP1 as a target protein in the preparation of anti-influenza virus drugs.
2. Use of TNFAIP1 as a target protein in the preparation of an anti-influenza virus drug according to claim 1, characterized in that: The influenza virus is influenza A virus.
3. Use of TNFAIP1 as a target protein according to claim 1 or 2 in the preparation of an anti-influenza virus drug, characterized in that: The TNFAIP1 or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier and / or excipient to form any pharmaceutically acceptable dosage form.
4. Use of TNFAIP1 as a target protein in the preparation of an anti-influenza virus drug according to claim 3, characterized in that: The dosage forms include powder for injection, capsules, tablets, and suspensions.
5. Use of TNFAIP1 protein or a pharmaceutically acceptable salt thereof in the preparation of an adjuvant for influenza virus vaccine.
6. Use of the TNFAIP1 protein or a pharmaceutically acceptable salt thereof according to claim 5 in the preparation of an adjuvant for an influenza virus vaccine, characterized in that: The influenza virus is influenza A virus.
7. Use of the TNFAIP1 protein or a pharmaceutically acceptable salt thereof according to claim 5 or 6 in the preparation of an adjuvant for an influenza virus vaccine, characterized in that: The TNFAIP1 or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier and / or excipient to form any pharmaceutically acceptable dosage form.
8. Use of the TNFAIP1 protein or a pharmaceutically acceptable salt thereof according to claim 7 in the preparation of an adjuvant for an influenza virus vaccine, characterized in that: The dosage forms include oil-in-water emulsion adjuvant and aluminum adjuvant.