Application of tyrosine kinase LYN in anti-influenza virus
By adding tyrosine kinase LYN to the cells, the replication of influenza A virus is suppressed, and the problem of difficult to effectively inhibit influenza virus replication in the prior art is solved, and an effective method for preparing anti-influenza virus drugs or adjuvants is provided.
Patent Information
- Application Number
- CN202510252877.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-05
- Publication Date
- 2025-05-30
AI Technical Summary
The prior art is difficult to effectively inhibit the replication of influenza A virus, especially in pregnant women, the elderly, children and people with low immunity, resulting in severe respiratory diseases and death.
Anti-influenza virus drugs or adjuvants are prepared using LYN protein or pharmaceutically acceptable salts thereof by adding tyrosine kinase LYN to cultured cells.
LYN can significantly inhibit the replication of influenza A virus, providing a scientific basis and operating method for the preparation of anti-IAV infection drugs or adjuvants, and is of great significance and value.
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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 tyrosine kinase LYN in anti-influenza virus Background Art
[0002] Influenza A virus (IAV) belongs to the genus Influenzavirus A of the family Orthomyxoviridae. It is a single-stranded negative-strand segmented RNA virus that can infect various animals such as humans, birds, and pigs. It is the pathogen that has caused global influenza epidemics in recent decades. Although there are currently vaccines available for preventing influenza virus infection, due to the complex typing of influenza viruses and limitations in epidemic prediction capabilities, outbreaks still occur from time to time. Moreover, it can cause severe respiratory diseases and even death in pregnant women, the elderly, children, and some people with chronic diseases or low immunity. According to statistics, millions of people are infected with influenza viruses globally every year, and hundreds of thousands of people die from severe respiratory diseases related to influenza. Due to the high variability of the proteins encoded by influenza viruses, especially the hemagglutinin enzyme (HA) and neuraminidase (NA) on their surfaces, there are currently no very effective broad-spectrum antiviral drugs and influenza vaccines. Therefore, finding new targets for resisting influenza viruses is of great significance.
[0003] LYN (Lck / Yes-related novel protein tyrosine kinase) is a class of non-receptor protein tyrosine kinases belonging to the Src family. It can receive and transmit signals from cell surface receptors, play important roles in regulating proliferation, differentiation, apoptosis, migration, and metabolism, and play a regulatory role in the processes of innate immunity and adaptive immunity. Currently, research on LYN related to viruses mainly focuses on immunodeficiency viruses (HIV), tumor-related herpesviruses (EBV, KSHV), etc. Its main mode of action is that viral proteins regulate the kinase activity of LYN to activate or shut down various signal pathways downstream of LYN, thereby regulating the proliferation, apoptosis, metabolism, and immune activity of host cells.
[0004] The present invention discovers that adding LYN to cells culturing IAV can reduce the replication level of IAV, indicating that LYN has the effect of inhibiting the replication of IAV and can be used to prepare drugs or adjuvants for anti-IAV infection to inhibit the replication of IAV. Summary of the Invention
[0005] The object of the present invention is to provide the application of tyrosine kinase LYN in anti-influenza virus.
[0006] The technical solution of the present invention is: the application of tyrosine kinase LYN in anti-influenza virus.
[0007] As a further improvement of the present invention, the influenza virus is an influenza A virus.
[0008] As a further improvement of the present invention, LYN protein or a pharmaceutically acceptable salt thereof is used for preparing an anti-influenza virus drug.
[0009] As a further improvement of the present invention, LYN protein or a pharmaceutically acceptable salt thereof is added with a pharmaceutically acceptable carrier and / or excipient to form any pharmaceutically acceptable dosage form.
[0010] Furthermore, the dosage form includes powder for injection, capsules, tablets, and suspensions.
[0011] As a further improvement of the present invention, LYN protein or a pharmaceutically acceptable salt thereof is used for preparing a virus vaccine adjuvant.
[0012] As a further improvement of the present invention, LYN protein or a pharmaceutically acceptable salt thereof is added with a pharmaceutically acceptable carrier and / or excipient to form any pharmaceutically acceptable dosage form.
[0013] Furthermore, the dosage form includes oil-in-water emulsion adjuvant and aluminum adjuvant.
[0014] The beneficial effects of the present invention are as follows: adding LYN to the cells for culturing IAV can reduce the replication level of IAV, indicating that LYN has the effect of inhibiting the replication of IAV and can be used for preparing drugs or adjuvants against IAV infection to inhibit the replication of IAV. The present invention provides the application of LYN protein in anti-influenza virus, as well as specific application means and application objects, providing a strong scientific basis and operation methods for the prevention, control, and treatment of influenza A virus, and having great significance and value. Description of the Drawings
[0015] Figure 1 It is a result diagram of overexpressing LYN protein to inhibit IAV replication, where A is the virus titration result of IAV, and B is the Western Blot result; Figure 2 It is a result diagram of knocking down LYN to promote IAV replication, where A is the result of verifying the effect of si LYN, B is the result of measuring the IAV virus titer, and C is the qPCR result of the mRNA expression of LYN; Figure 3 It is a result diagram of the virus titration in the cell supernatant after overexpressing LYN protein to inhibit the infection of different subtypes of IAV; Figure 4 It is a result diagram of knocking out LYN to promote IAV replication in mice, where A is the result of the mouse body weight change curve, B is the result of the mouse survival curve, and C is the result of organ titration. Detailed Embodiments
[0016] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.
[0017] The experimental methods in the following examples are all conventional methods unless otherwise specified; the test materials used in the following examples are all obtained from regular biochemical reagent companies unless otherwise specified.
[0018] The LYN protein expression plasmid was constructed by the Innovative Team of Animal Virus Molecular Ecology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The amino acid sequence of the LYN protein is shown in SEQ ID NO.1. Since the sequence contains nucleotides not included in Table 1 of the "Standard for Nucleotide and / or Amino Acid Sequence List and Sequence List Electronic File", the S26 standard nucleotide or amino acid sequence computer-readable carrier is not provided.
[0019] SEQ ID NO.1: MGCIKSKGKDSLSDDGVDLKTQPVRNTERTIYVRDPTSNKQQRPVPESQLLPGQRFQTKDPEEQGDIVVALYPYDGIHPDDLSFKKGEKMKVLEEHGEWWKAKSLLTKKEGFIPSNYVAKLNTLETEEWFFKDITRKDAERQLLAPGNSAGAFLIRESETLKGSFSLSVRDFDPVHGDVIKHYKIRSLDNGGYYISPRITFPCISDMIKHYQKQADGLCRRLEKACISPKPQKPWDKDAWEIPRESIKLVKRLGAGQFGEVWMGYYNNSTKVAVKTLKPGTMSVQAFLEEANLMKTLQHDKLVRLYAVVTREEPIYIITEYMAKGSLLDFLKSDEGGKVLLPKLIDFSAQIAEGMAYIERKNYIHRDLRAANVLVSESLMCKIADFGLARVIEDNEYTAREGAKFPIKWTAPEAINFGCFTIKSDVWSFGILLYEIVTYGKIPYPGRTNADVMTALSQGYRMPRVENCPDELYDIMKMCWKEKAEERPTFDYLQSVLDDFYTATEGQYQQQP.
[0020] Cell culture: A549 cells are derived from human lung cancer epithelial cells; they are cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% double antibiotics in an incubator with 5% CO 2 at 37 °C.
[0021] Virus source: The IAV-PR8 strain is stored in the Innovative Team of Animal Virus Molecular Ecology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.
[0022] Example 1: LYN protein inhibits IAV virus replication.
[0023] 1. Transfect the LYN plasmid into A549 cells In A549 cells, transfect the LYN plasmid at a concentration of 1 μg / mL.
[0024] 2. Preparation of samples for incubating cells infected with IAV with LYN protein Twenty hours after transfection, inoculate the IAV-PR8 strain (MOI = 0.01). After inoculation, collect the cell supernatant and cells at 12 h, 24 h, 48 h, and 72 h respectively.
[0025] 3. Determination of IAV virus titer Determine the EID of the supernatant obtained in step 2 50 , and perform virus titer analysis.
[0026] EID 50 The determination steps are as follows: Prepare SPF chicken embryos at 9 - 11 days old. Inoculate the supernatant collected in step 2 and make 10-fold serial dilutions until 10 -8 , select 10 -2 -10 -8 , inoculate 9 - 11-day-old SPF chicken embryos, three chicken embryos for each dilution, 0.1 mL / embryo. Incubate in an incubator at 37 After 48 h of incubation, take out all the chicken embryos for hemagglutination detection. If there is hemagglutination, it is judged as positive for infection. Then, according to the hemagglutination titer formula, calculate the EID 50 , which is the virus titer.
[0027] The virus titration results are as shown in Figure 1 A. The LYN protein significantly reduced the titer of IAV in the A549 cell supernatant and inhibited IAV replication.
[0028] 4. Detection of the content of IAV-NP protein Collect cell proteins 48 h after infection and determine the content of NP protein by Western Blot.
[0029] The experimental results are as shown in Figure 1 B. After adding the LYN plasmid, the expression level of the IAV-NP protein was significantly reduced, indicating that overexpression of the LYN protein significantly inhibited the expression of the IAV-NP protein compared with normal A549 cells.
[0030] In summary, the above experiments showed that, compared with normal A549 cells, overexpression of LYN protein significantly inhibited IAV virus replication. Therefore, overexpression of LYN protein or LYN protein expression promoter by transfection or other means can significantly inhibit IAV virus replication and can be used for the prevention or treatment of IAV infection.
[0031] Example 2: Knockdown of LYN protein promotes IAV replication.
[0032] 1. Select effective LYN siRNA The synthesized siRNA targeting LYN was reverse transfected into A549 cells. Cell proteins were collected 24 h after transfection, and then the LYN protein content was determined by Western Blot. The experimental results were as Figure 2 shown in B. Compared with NC, si-LYN had a highly significant knockdown effect at the protein level.
[0033] 2. Determination of IAV virus titer The detection method was the same as that in Example 1.
[0034] The virus titration results were as Figure 2 shown in A. Downregulation of LYN protein expression significantly increased the EID of IAV in A549 cells 50 , promoting IAV replication.
[0035] 3. qPCR detection of IAV mRNA expression level Cells were collected at 0 h, 12 h, and 24 h after infection. Total RNA was extracted according to the instruction manual of the TAKARA RNAiso Plus total RNA extraction kit and reverse transcribed. The obtained cDNA was subjected to real-time fluorescence quantitative PCR detection to compare the differences in virus mRNA levels between different groups.
[0036] The experimental results were as Figure 2 shown in C. The addition of LYN siRNA significantly increased the IAV mRNA expression level, and with the increase of IAV infection time, the IAV mRNA expression level after the addition of LYN siRNA was more significantly increased compared with NC siRNA, indicating that the replication of IAV increased after inhibiting LYN protein expression.
[0037] The above results showed that after downregulating the expression level of LYN protein, IAV replication could be promoted. Therefore, LYN inhibitor can be used to inhibit LYN protein expression and promote IAV replication.
[0038] Example 3: LYN inhibits the replication of different subtypes of IAV virus.
[0039] The detection method was the same as that in Example 1.
[0040] The experimental results were asFigure 3 As shown, LYN protein significantly reduced the titers of different subtypes of IAV in the supernatant of A549 cells and inhibited the replication of different subtypes of IAV.
[0041] Example 4: LYN knockout promotes IAV replication in mice.
[0042] 1. Determination of the median lethal dose (MLD Lyn + / + and Lyn - / - in mice 50 ) Prepare 30 six-week-old female C57BL6 / N Lyn + / + and Lyn - / - mice, 5 in each group, 3 days before virus challenge. Take the same PFU of IAV and make serial 10-fold dilutions to 10. After dilution, place it on ice for later use. After anesthesia with inhaled CO 2 , each mouse was inoculated nasally with virus (50 µl) or PBS. Observe the status of the mice every day after virus challenge, weigh the mice, and record the death situation (when the weight drops by 25%, it is recorded as death).
[0043] The experimental results are as shown in Figure 4 A and 4B. Compared with Lyn + / + mice, Lyn - / - showed faster weight loss and death after infection with the same dose of virus. According to their death situations, we calculated the MLD of influenza virus in the two types of mice respectively 50 , Lyn + / + The MLD of 50 mice 10 = 2.68 Log 50 EID - / - The MLD of Lyn 50 <0.83 Log 10 EID 50 .
[0044] 2. Virus titers in the organs of virus-challenged mice On the 5th day after infection, 3 mice in each group were euthanized, and the virus amounts in their nasal turbinates, lungs, brains, livers, and kidneys were examined. The detection method was the same as in Example 1.
[0045] The experimental results are as shown in Figure 4 C. Compared with Lyn + / + mice, Lyn - / -Promote the replication of IAV in different organs of mice.
[0046] In summary, taking host cell A549 cells and mice as examples in the above embodiments, it was studied that LYN can inhibit the replication of IAV in vitro and in vivo, indicating that LYN can inhibit the replication of influenza A virus and can be used to prepare drugs or adjuvants against influenza A virus infection.
[0047] It should be noted that taking different subtypes of influenza virus as examples, the present invention proves that LYN can inhibit the replication of different subtypes of influenza virus and can be used to prepare drugs or adjuvants against influenza A virus infection.
[0048] The present invention found that overexpression of the LYN gene can significantly reduce the expression of influenza A virus protein, viral mRNA and virus titer, and significantly inhibit the replication of influenza A virus; in addition, after knocking down / knocking out the LYN protein, the expression of influenza A virus mRNA and the replication level of the virus in mice were increased, indicating that LYN has the effect of inhibiting the replication of influenza A virus in vitro and in vivo and can be used to prepare drugs or adjuvants against influenza A virus infection.
Claims
1. Application of tyrosine kinase LYN in anti-influenza virus.
2. The use of tyrosine kinase LYN in anti-influenza A virus according to claim 1, characterized in that: The influenza virus is influenza A virus.
3. The use of tyrosine kinase LYN in anti-influenza A virus according to claim 1 or 2, characterized in that: The LYN protein or a pharmaceutically acceptable salt thereof is used for preparing anti-influenza virus drugs.
4. The use of tyrosine kinase LYN in anti-influenza A virus according to claim 3, characterized in that: The LYN protein or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier and / or excipient to prepare any pharmaceutically acceptable dosage form.
5. The use of tyrosine kinase LYN in anti-influenza A virus according to claim 4, characterized in that: The dosage forms include powder injection, capsule, tablet and suspension.
6. Use of the tyrosine kinase LYN in resisting influenza A virus according to claim 1 or 2, characterized in that: The LYN protein or a pharmaceutically acceptable salt thereof is used for preparing a virus vaccine adjuvant.
7. The use of tyrosine kinase LYN in anti-influenza A virus according to claim 6, characterized in that: The LYN protein or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier and / or excipient to prepare any pharmaceutically acceptable dosage form.
8. The use of tyrosine kinase LYN in resisting influenza A virus according to claim 7, characterized in that: The dosage forms include oil emulsion adjuvant and aluminum adjuvant.