Use of lgals4 gene in anti-influenza virus

Abstract

The application belongs to the field of genetic engineering and biological medicine technology, and particularly relates to application of LGALS4 gene in resisting influenza virus. The application first finds that the LGALS4 gene can be used as a key target point for regulating H3 AIV replication, that is, inhibiting the LGALS4 gene can significantly inhibit the replication of H3 AIV, and then inhibit the infection of H3 AIV, and overexpressing the LGALS4 gene can effectively promote the replication of H3 AIV and improve the virus titer. The research provides a brand-new action mechanism for an antiviral strategy.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and biomedicine, specifically relating to the application of the LGALS4 gene in anti-influenza virus. Background Technology

[0002] Influenza viruses are significant pathogens that seriously threaten human and animal health. They can spread between humans and animals, causing acute respiratory illnesses and leading to seasonal epidemics or even pandemics. The H3 subtype of avian influenza virus (IAV) is one of the important subtypes of influenza A virus. H3 AIV is not only widespread in waterfowl such as ducks and geese, but can also infect mammals, posing a potential threat to poultry farming and public health. Currently, the treatment of H3 subtype avian influenza virus revolves around early use of antiviral drugs combined with supportive care. However, due to the continuous recombination and mutation of influenza viruses, increasing drug resistance, difficulties in finding drug targets, limited broad-spectrum and time-sensitive vaccines, and significant bottlenecks in aerosol transmission blocking technology, the effectiveness of traditional vaccines and antiviral drugs is becoming increasingly limited.

[0003] Therefore, there is an urgent need to develop a new method to effectively regulate the resistance of influenza viruses. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to provide the application of the LGALS4 gene in anti-influenza virus.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides the application of the LGALS4 gene in regulating host antiviral activity, wherein the virus is an H3 subtype avian influenza virus.

[0006] A second aspect of the present invention provides the use of a substance that inhibits LGALS4 gene expression in the preparation of a drug for treating H3 subtype avian influenza virus.

[0007] A third aspect of the present invention provides the use of a substance that inhibits LGALS4 gene expression in the in vitro inhibition of pathogen proliferation in cells infected with H3 subtype avian influenza virus.

[0008] In a fourth aspect, the invention provides the use of a substance that inhibits LGALS4 gene expression in the in vitro inhibition of pathogen protein production in cells infected with H3 subtype avian influenza virus.

[0009] In a fifth aspect, the present invention provides a drug for combating H3 subtype avian influenza virus, characterized in that the drug contains a substance that inhibits the expression of the LGALS4 gene.

[0010] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: This invention reveals for the first time that the LGALS4 gene is a key target for regulating H3 AIV replication. Experiments demonstrate that overexpression of LGALS4 significantly promotes viral replication, thereby increasing viral titer, while knockdown of LGALS4 effectively inhibits viral replication, thereby reducing viral titer, providing a novel mechanism of action for antiviral strategies. Specifically, biomaterials based on LGALS4 overexpression can efficiently enhance the replication efficiency of H3 AIV in host cells, overcoming the technical bottleneck of insufficient viral titer in virus culture and providing high-titer viral raw materials for vaccine production and virological research. Conversely, biomaterials based on LGALS4 knockdown can effectively block H3 AIV viral replication, reducing infection rates and providing a new candidate target for developing anti-influenza drugs, thus possessing significant practical application value. Attached Figure Description

[0011] Figure 1 To detect the knockdown rate of LGALS4-shRNA.

[0012] Figure 2 The fluorescence expression and mRNA expression of LGALS4 in the A549 cell line were investigated to stabilize LGALS4.

[0013] Figure 3 To investigate the effect of LGALS4 knockdown on the viral titer of H3 AIV in A549 cell lines.

[0014] Figure 4 The changes in LGALS4 gene levels after transient transfection of A549 cells.

[0015] Figure 5 To investigate the effect of overexpression of the LGALS4 gene on the viral titer of H3 AIV. Detailed Implementation

[0016] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration 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.

[0017] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. 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, and / or combinations thereof.

[0018] In this invention, lectin galctoside-binding soluble 4 (LGALS4), located on human chromosome 19q13.2, is an important member of the galctoside family. It is a class of soluble sugar-binding proteins primarily expressed in intestinal epithelial tissue, participating in various biological processes by specifically recognizing and binding to β-galactosidic sugar ligands. The human LGALS4 gene has a Gene ID of 3960 on the NCBI website.

[0019] In a typical embodiment of the present invention, the application of the LGALS4 gene in regulating host antiviral activity is provided, wherein the virus is an H3 subtype avian influenza virus.

[0020] In some embodiments, the regulation is as follows: inhibiting LGALS4 gene expression can enhance the host's resistance to infection and inhibit the proliferation of H3 subtype avian influenza virus, while overexpressing LGALS4 gene makes the host susceptible to H3 subtype avian influenza virus.

[0021] Furthermore, substances that inhibit LGALS4 gene expression can be used to prepare influenza virus inhibitors. Biomaterials that promote LGALS4 gene expression can be used to prepare influenza virus promoters.

[0022] Specifically, the influenza virus inhibitor inhibits the proliferation and replication of H3 AIV, thereby significantly suppressing H3 AIV infection. The influenza virus promoter can efficiently enhance the replication efficiency of H3 AIV in host cells, overcoming the technical bottleneck of insufficient titer in virus culture and providing high-titer viral raw materials for vaccine production and virological research.

[0023] In another typical embodiment of the present invention, the use of the substance that inhibits LGALS4 gene expression is provided in any of the following: (1) Used in the preparation of drugs against H3 subtype avian influenza virus; (2) Used to inhibit the proliferation of pathogens in cells infected with H3 subtype avian influenza virus in vitro; (3) Used to inhibit the production of pathogen proteins in cells infected with H3 subtype avian influenza virus in vitro.

[0024] In some embodiments, the substance that inhibits LGALS4 gene expression includes RNA interference molecules or antisense oligonucleotides, small molecule inhibitors, siRNA, shRNA, substances that induce lentiviral infection or gene knockout, and specific antibodies against the substance itself or its upstream or downstream molecules.

[0025] In some implementations, LGALS4 gene expression is suppressed by designing shRNAs that target the LGALS4 gene.

[0026] Specifically, lentiviral vectors are used to integrate the shRNA gene into the host genome, achieving stable knockdown that can last for weeks or even longer.

[0027] In this invention, only one set of shRNAs targeting the LGALS4 gene is provided as an example. To comprehensively cover the target gene's action site and improve the stability and reliability of gene silencing effects, a series of shRNAs can be specifically designed to completely cover the entire sequence of the LGALS4 gene. By targeting different regions of the gene, efficient inhibition of LGALS4 gene expression can be achieved, avoiding the problem of inhibition failure caused by mutations at a single target site. Furthermore, those skilled in the art can design corresponding series of shRNAs based on the nucleotide sequence characteristics of the LGALS4 gene in different species, and the shRNAs corresponding to each species can completely cover the entire sequence of their own LGALS4 gene, ensuring specific targeting effects on the LGALS4 gene in different species.

[0028] In some embodiments, the shRNA comprises a sense strand and an antisense strand of a DNA oligo, wherein the sense strand has a nucleotide sequence as shown in any one of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0029] In some embodiments, the pathogen protein includes at least one of nucleoprotein, hemagglutinin, neuraminidase, RNA polymerase basic protein, matrix protein, and non-structural high protein.

[0030] In another typical embodiment of the present invention, an anti-H3 subtype avian influenza virus drug is provided, characterized in that the drug contains a substance that inhibits the expression of the LGALS4 gene.

[0031] In some embodiments, the substance that inhibits LGALS4 gene expression includes RNA interference molecules or antisense oligonucleotides, small molecule inhibitors, siRNA, shRNA, substances that induce lentiviral infection or gene knockout, and specific antibodies against the substance itself or its upstream or downstream molecules.

[0032] In some embodiments, the shRNA comprises a sense strand and an antisense strand of a DNA oligo, wherein the sense strand has a nucleotide sequence as shown in any one of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0033] In this embodiment, the pLenti-U6-shRNA-CMV-copGFP-P2A-PURO vector was purchased from WZ Biosciences Inc., catalog number 20251127008; the overexpression plasmid Penter-LGALS4-6his1flag was constructed by WZ Biosciences Inc., catalog number CH833172, and can also be constructed by those skilled in the art, as long as the LGALS4 gene is overexpressed; the helper plasmids psPAX2 and pMD2.G were preserved by the Institute of Animal Husbandry and Veterinary Medicine, Shandong Academy of Agricultural Sciences, and are available to the public from the Institute of Animal Husbandry and Veterinary Medicine, Shandong Academy of Agricultural Sciences, and are only used for replicating this invention and not for other commercial purposes.

[0034] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0035] Example 1 1. Design of shRNA targeting LGALS4 Using the LGALS4 gene as a template (Gene ID: 3960), a shRNA targeting human LGALS4 was synthesized by Shandong Weizhen Biotechnology Co., Ltd., with a nonsense sequence as a control. It was named LGALS4-shNC. The sense strand of the shRNA sequence is shown in Table 1.

[0036] Table 1. shRNA sequences targeting human LGALS4

[0037] 2. Construction of a recombinant lentiviral expression plasmid that specifically inhibits LGALS4 gene expression Based on the shRNA forward and reverse strand sequences in Table 1, the sequences of the sense and antisense strands were further synthesized, and the sense and antisense strands were annealed and inserted into the BamHI / MluI restriction sites of the pLenti-U6-shRNA-CMV-copGFP-P2A-PURO vector (purchased from WZ Biosciences Inc., catalog number: 20251127008) to construct lentiviral plasmids LGALS4-sh1, LGALS4-sh2, LGALS4-sh3 and control group LGALS4-shNC. These plasmids were transformed into DH5α competent cells, and after culturing the resulting single colonies for 12 hours, they were sent for sequencing. Positive clones that were correctly identified by sequencing were cultured overnight (12-16 hours) in 25-35 mL LB (containing ampicillin), and then the target plasmids were extracted using an endotoxin-free / small-volume extraction kit.

[0038] Example 2 Construction of an A549 cell line that specifically inhibits LGALS4 gene expression.

[0039] 1. Detection of LGALS4-shRNA knockdown rate LGALS4 knockdown plasmids LGALS4-sh1, LGALS4-sh2, LGALS4-sh3, and the control group LGALS4-shNC were transfected into HEK-293T cells at a 1:10 ratio using Lipofectamine™ 2000. Cells were collected 72 h later, and LGALS4 expression levels were measured by qRT-PCR. Results are as follows: Figure 1 As shown, all three shRNAs can reduce the expression level of LGALS4 mRNA to varying degrees, with LGALS4-sh3 exhibiting the highest knockdown efficiency, which will be used for subsequent lentiviral packaging.

[0040] 2. LGALS4-sh3 knockdown plasmid was transfected into 293T cells to produce lentivirus. HEK-293T cells were cultured, and when the cells reached approximately 80% confluency, the knockdown plasmid LGALS4-sh3 and helper plasmids psPAX2 and pMD2.G were transfected into HEK-293T cells using Lipofectamine™ 2000 at a mass ratio of 2:2:1. The shNC plasmid was used as a control group. After 24 h, 5 mL of culture medium was collected (stored at 4°C), and 5–6 mL of culture medium containing 10% serum-conjugated antibiotics was added.

[0041] After 48 h, collect 5 mL of supernatant and add 5–6 mL of DMEM medium containing 10% serum and penicillin antibodies. After 72 h, collect all supernatants. Centrifuge all collected supernatants at 3000 rpm, 4 °C for 15 min and filter through a 0.45 µm filter membrane. Add 8–10 μg / mL polybrene (1000×), aliquot, and store at -80 °C.

[0042] 3. Lentiviral infection of A549 cells A549 cells were seeded in 6-well plates and, when they reached approximately 80% confluence, packaged lentivirus (MOI=5) was added. The medium was changed after 48 hours, and 1 μg / mL of puromycin was added for selection. A stable cell line was formed in about one week.

[0043] 4. Identification of A549 cell lines that specifically inhibit LGALS4 gene expression Cells from stable cell lines were collected, and their expression was observed under a fluorescence microscope. Simultaneously, stable cell lines with silenced LGALS4 and the shNC control group were collected, and the expression level of LGALS4 mRNA was detected by qRT-PCR. Results are as follows: Figure 2As shown, the stable knockdown cell line exhibits green fluorescence under a fluorescence microscope, and the expression level of the LGALS4 gene in the knockdown cell line is significantly reduced, indicating that a stable LGALS4 knockdown cell line and a control cell line have been successfully constructed.

[0044] Example 3 Effect of A549 cell line with specific inhibition of LGALS4 gene expression on H3 AIV proliferation.

[0045] Cells with stably silenced LGALS4 gene and shNC control cells were cultured to approximately 80% confluence, then infected with H3 subtype AIV (MOI=0.1). After 24 h of infection at 37°C in a 5% CO2 incubator, the supernatant was collected. The collected supernatant was serially diluted 10-fold. -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 10 -9 10 -10 The viral titer of H3 subtype AIV was detected by hemagglutination assay after culturing 96-well plates of MDCK monolayer cells for 60 h. Results are as follows: Figure 3 As shown, the results indicate that specifically inhibiting LGALS4 gene expression can significantly suppress the viral titer of H3 AIV.

[0046] Example 4 Construction of expression plasmids for overexpressing the LGALS4 gene.

[0047] The human LGALS4 gene (Gene ID: 3960) was cloned into the Penter vector, with a 6his1flag tag added to the C-terminus. The restriction enzyme sites were AsisI / MluI, and the vector was named Penter-LGALS4-6his1flag. Positive clones with correct sequencing were cultured overnight (13-16 h) in 25-35 mL LB (Kan+) solution, and high-purity plasmids were extracted using a plasmid extraction kit for later use.

[0048] Example 5 The effect of overexpression of the LGALS4 gene on H3 AIV.

[0049] 1. Transient transfection of A549 cells with overexpression of the LGALS4 gene The empty vector plasmid Penter-6his1flag (OE-NC group) and the overexpression plasmid Penter-LGALS4-6his1flag (OE-LGALS4 group) were transfected into A549 cells with Lipofectamine 2000. Cells were collected 24 h after transfection, and the expression level of the LGALS4 gene was verified by qRT-PCR.

[0050] The results are as follows Figure 4 As shown in the results, the expression level of LGALS4 in the OE group was significantly higher than that in the OE-NC group, indicating that A549 cells were successfully transiently transfected with an overexpression of the LGALS4 gene.

[0051] 2. Functional differences in H3 subtype AIV cells after infection. A549 cells from the OE-NC and OE-LGALS4 groups, transfected for 24 h, were infected with H3 subtype AIV (MOI=0.1). Supernatants were collected 24 h after infection and used to detect H3 subtype AIV TCID. 50 .

[0052] The collected supernatant was serially diluted 10-fold, with each dilution being 10... -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 10 -9 10 -10 The viral titer of H3 subtype AIV was detected by hemagglutination assay after culturing 96-well plates of MDCK monolayer cells for 60 h. Results are as follows: Figure 5 As shown, the results indicate that overexpression of the LGALS4 gene can significantly promote the viral titer of H3 AIV.

[0053] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of the LGALS4 gene in regulating host antiviral activity, characterized in that, The virus in question is the H3 subtype of avian influenza virus.

2. The application as described in claim 1, characterized in that, The regulation is as follows: inhibiting LGALS4 gene expression can enhance the host's resistance to infection and inhibit the proliferation of H3 subtype avian influenza virus, while overexpressing LGALS4 gene makes the host susceptible to H3 subtype avian influenza virus.

3. Application of substances that inhibit LGALS4 gene expression in the preparation of drugs against H3 subtype avian influenza virus.

4. Application of substances that inhibit LGALS4 gene expression in inhibiting pathogen proliferation in cells infected with H3 subtype avian influenza virus in vitro.

5. Application of substances that inhibit LGALS4 gene expression in the in vitro inhibition of pathogen protein production in cells infected with H3 subtype avian influenza virus.

6. The application as described in claim 5, characterized in that, The pathogen proteins include at least one of the following: nucleoprotein, hemagglutinin, neuraminidase, RNA polymerase, basic protein, matrix protein, and non-structural high protein.

7. The application as described in any one of claims 3-5, characterized in that, The substances that inhibit LGALS4 gene expression include RNA interference molecules or antisense oligonucleotides, small molecule inhibitors, siRNA, shRNA, substances that induce lentiviral infection or gene knockout, and specific antibodies against the gene itself or its upstream and downstream molecules.

8. The application as described in claim 7, characterized in that, The shRNA comprises a sense strand and an antisense strand of a DNA oligo, wherein the sense strand has a nucleotide sequence as shown in any one of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:

3.

9. A drug for treating H3 subtype avian influenza virus, characterized in that, The drug contains a substance that inhibits the expression of the LGALS4 gene.

10. The anti-H3 subtype avian influenza virus drug as described in claim 9, characterized in that, The substances that inhibit LGALS4 gene expression include RNA interference molecules or antisense oligonucleotides, small molecule inhibitors, siRNA, shRNA, substances that induce lentiviral infection or gene knockout, and specific antibodies against the gene itself or its upstream and downstream molecules.

Images