A lncRNA and its encoded polypeptide and application

Abstract

The present invention discloses a lncRNA, its encoded polypeptide, and its application. Experiments have confirmed that silencing the lncRNA of the present invention can significantly inhibit viral replication; overexpressing the lncRNA of the present invention in cells can significantly promote viral replication after infection with different subtypes of influenza viruses. The lncRNA of the present invention and its polypeptide GVRP1-ORF play an important role in the influenza virus replication process, providing new ideas and strategies for screening drugs for treating and / or preventing various influenza viruses, and providing a new target for the treatment of influenza virus infection, with broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a lncRNA, its encoded polypeptide and application. Background Art

[0002] Influenza A virus (IAV) is one of the most common causes of respiratory illness in humans. Despite the use of vaccines to control H9N2 avian influenza, the virus continues to predominate, and the underlying causes remain unknown. Furthermore, in recent years, H9N2 avian influenza virus infections in humans have continued to occur, posing a threat to human health. However, the molecular mechanisms of cross-species transmission and the regulatory mechanisms involving host factors remain largely unresolved.

[0003] Non-coding RNAs (ncRNAs) are a class of RNA transcripts transcribed from the genome. At the RNA level, they can exert their respective biological functions and play a crucial role in processes such as cell growth, differentiation, replication, and apoptosis. Long non-coding RNAs (lncRNAs), a subclass of ncRNAs, are longer than 200 nt and are transcribed by RNA polymerase II. They are cleaved to form a 5' cap structure and a 3' poly A tail. lncRNAs possess one or more short open reading frames and have the potential to encode functional micropeptides. These functional micropeptides play a crucial role in regulating body homeostasis and in the development and progression of diseases and cancer.

[0004] In recent years, research on lncRNAs and their encoded polypeptides has primarily focused on human diseases and cancer. Peptides primarily influence cancer progression by acting as oncogenic factors or tumor suppressors. Influenza virus infection can induce significant changes in the expression of many host factors. Some host factors can directly target viral genes and affect viral replication during infection, or indirectly inhibit IAV replication by regulating intracellular signaling. lncRNAs can interact with a variety of biomacromolecules and may play an important role in regulating influenza virus replication, either directly or indirectly. Therefore, this patent combines bioinformatics analysis and experimental verification to identify a novel long non-coding RNA and its encoded micropeptide, and explores its application in drug detection for influenza virus infection, providing a new approach for the design of anti-influenza drugs. Summary of the Invention

[0005] In view of the shortcomings of the existing problems, the purpose of the present invention is to provide a lncRNA and its encoded polypeptide and application.

[0006] The technical solution adopted by the present invention to solve the technical problem is:

[0007] In a first aspect, the present invention provides an unreported functional avian lncRNA and the polypeptide encoded thereby.

[0008] In the early stage of the present invention, whole transcriptome sequencing was performed after host cells were infected with the H9N2 avian influenza strain, and combined with data analysis, a novel lncRNA that regulates influenza virus replication was discovered: lncRNA GVRP1; the lncRNA gene ID is ENSGALT00000106466, and the gene is located at chr7: 9980172-9982804; its nucleotide sequence is shown in SEQ ID No. 1; the transcribed RNA sequence is shown in SEQ ID No. 2.

[0009] The translated polypeptide GVRP1-ORF was specifically expressed in DF1 cells. Western blot and indirect immunofluorescence confirmed that 225 bases of lncRNA GVRP1 could be transcribed and translated into polypeptide. The amino acid sequence of GVRP1-ORF is shown in SEQ ID NO.3.

[0010] In a second aspect, the present invention also protects a vector containing the lncRNA described above.

[0011] In a specific embodiment, the vector can be a plasmid, a lentiviral vector, or the like.

[0012] In a third aspect, the present invention also protects the use of the aforementioned lncRNA, the aforementioned polypeptide, or the aforementioned vector in promoting the proliferation of influenza viruses. For example, by promoting the proliferation of influenza viruses, a cell model of highly expressed influenza viruses can be constructed, thereby screening candidate substances for preventing and treating influenza viruses.

[0013] In a fourth aspect, the present invention protects a drug for detecting influenza virus infection, including a reagent material that encodes a polypeptide encoded by a lncRNA or promotes the expression of the lncRNA;

[0014] The lncRNA is lncRNA GVRP1, the nucleotide sequence of the lncRNA GVRP1 is shown as SEQ ID NO. 1; the amino acid sequence of the polypeptide GVRP1-ORF encoded by the lncRNA GVRP1 is shown as SEQ ID NO. 3; the reagents for promoting lncRNA expression include plasmids and lentiviral vectors carrying the cDNA sequence of the lncRNA GVRP1 polypeptide GVRP1-ORF, and GVRP1-ORF mouse polyclonal antibodies, etc.

[0015] In a specific embodiment, the drug comprises a specific primer pair for detecting the level of lncRNA in a test sample.

[0016] In a more specific embodiment, the primer pair sequences are shown as SEQ ID No. 4~7.

[0017] In a fifth aspect, the present invention also protects the lncRNA inhibitor described above, wherein the inhibitor is si-lncRNA, and its sequence is any one of SEQ ID NOs: 8 to 9.

[0018] In a sixth aspect, the present invention protects a pharmaceutical composition for preventing and treating influenza virus, wherein the pharmaceutical composition contains the inhibitor described above.

[0019] In a seventh aspect, the present invention protects the use of the aforementioned lncRNA inhibitor or the aforementioned pharmaceutical composition in the preparation of a medicament for preventing and treating influenza virus.

[0020] In a specific embodiment, the medicament further comprises a pharmaceutically acceptable carrier.

[0021] In specific embodiments, the inhibitor comprises a nucleic acid molecule, a small molecule chemical drug, or a nucleic acid construct.

[0022] In specific embodiments, the nucleic acid molecule comprises siRNA, shRNA, dsRNA, etc.

[0023] In a more specific embodiment, said inhibitor is siRNA.

[0024] In a more specific embodiment, the sequence is any one of SEQ ID NOs: 8 to 9.

[0025] In an eighth aspect, the present invention protects a method for inhibiting influenza virus proliferation in vitro, comprising administering the lncRNA inhibitor described above.

[0026] In specific embodiments, the inhibitor comprises a nucleic acid molecule, a small molecule chemical drug, or a nucleic acid construct.

[0027] In specific embodiments, the nucleic acid molecule comprises siRNA, shRNA, dsRNA, etc.

[0028] In a more specific embodiment, said inhibitor is siRNA.

[0029] In a more specific embodiment, the sequence is any one of SEQ ID NOs: 8 to 9.

[0030] The method is not intended for disease diagnosis and treatment.

[0031] In specific embodiments, the virus includes but is not limited to influenza A virus.

[0032] Beneficial effects

[0033] The lncRNA molecule, its encoded polypeptide, and its application provided by the present invention have the following beneficial effects compared with the prior art:

[0034] This study used whole-transcriptome sequencing analysis of DF1 cells infected with the H9N2 subtype influenza virus as a sample, identifying a novel lncRNA molecule, designated lncRNA GVRP1, with the nucleotide sequence shown in SEQ ID No. 1. This study discloses for the first time the novel use of lncRNA GVRP1 as a positive regulatory factor in influenza virus infection. Infection with influenza virus (IAV) specifically promotes the expression of lncRNA GVRP1 and its encoded polypeptide, GVRP1-ORF.

[0035] The present invention experimentally demonstrates that silencing lncRNA GVRP1 significantly inhibits viral replication; overexpressing lncRNA GVRP1 in cells significantly promotes viral replication after infection with different influenza virus subtypes. This invention reveals that lncRNA GVRP1 and its encoded polypeptide, GVRP1-ORF, play a crucial role in influenza virus replication, providing new insights and strategies for screening drugs for treating and / or preventing various influenza viruses, offering a new target for the treatment of influenza virus infections and promising broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 : Volcano plot of lncRNAs enriched in transcriptome sequencing data with significant expression differences.

[0037] Figure 2 : qRT-PCR detection of lncRNA GVRP1 expression in DF1 cells after virus infection; Figure 2 Panel A in the figure shows that DF1 cells were infected with H9N2, H3N2, and WSN influenza viruses at an MOI of 1 for 24 hours, and lncRNA GVRP1 expression was detected by qRT-PCR; Figure 2 Panel B: different time of virus infection (0, 6h, 12h, 24h, 36h); Figure 2 Panel C: cells were infected with viruses at different MOIs (0, 0.01, 0.1, 1, and 3).

[0038] Figure 3 :The results of lncRNA GVRP1 on viral replication. Figure 3 Panel A: qRT-PCR detection of the overexpression effect of lncRNA GVRP1 by transfection of GVRP1-pcDNA3.1 plasmid; Figure 3Panel B: Effect of overexpression of lncRNA GVRP1 on H9N2 virus replication in plaque assay; Figure 3 Figure C: Plaque assay showing the effects of overexpression of lncRNA GVRP1 on WSN virus replication; Figure 3 Figure D: Plaque assay showing the effects of overexpression of lncRNA GVRP1 on H3N2 virus replication; Figure 3 Panel E: qRT-PCR detection of the inhibitory effect of siRNA on the expression of lncRNA GVRP1; Figure 3 Figure F: Effect of silencing lncRNA GVRP1 expression on H9N2 virus replication using plaque assay.

[0039] Figure 4 :GVRP1-ORF basic biological characteristics diagram. Among them, Figure 4 Figure A: Western blot detection of exogenous protein expression; Figure 4 Panel B: Fluorescence detection of GVRP1-ORF signal peptide translation activity; Figure 4 Figure C: Alphafold3 simulated 3D conformation of GVRP1-ORF protein; Figure 4 Figure D: Localization of GVRP1-ORF in cells;

[0040] Figure 5 :GVRP1-ORF stable transfection cell line construction results. Among them, Figure 5 Panel A: Overexpression efficiency of lentiviral plasmid; Figure 5 Middle panel B: Western blot detection of endogenous expression of GVRP1-ORF in overexpression cell lines; Figure 5 Panel C: CCK8 detection of cell activity; Figure 5 Figure D: Plaque results of H9N2 virus replication in overexpression cell lines; Figure 5 Panel E: NP gene mRNA level; Figure 5 Figure F: NP protein expression results.

[0041] Figure 6 :lncRNA GVRP1 plays a major role in regulating viral replication. Figure 6 Figure A: Schematic diagram of lncRNAGVRP1 truncation; Figure 6 Panel B: Effects of overexpression of lncRNA GVRP1 and its truncations on H9N2 virus replication.

[0042] Figure 7 : Sankey diagram of KEGG enrichment analysis of genes co-expressed with lncRNA GVRP1. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. The specific examples are only used to explain the present invention and are not to be construed as limitations on the present invention. Those of ordinary skill in the art will appreciate that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purpose of the present invention, and that the scope of the present invention is defined by the claims and their equivalents. The experimental methods for which specific conditions are not specified in the following examples are generally tested under conventional conditions or under conditions recommended by the manufacturer. The reagents, biological materials, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0044] The H9N2 avian influenza virus strain A / chicken / Anhui / LH99 / 2017, WSN (H1N1), and H3N2 strains were maintained in our laboratory. DF-1 cells were purchased from the American Type Culture Collection (ATCC) (Manassas, VA). The pLOX-IRES-TK lentiviral overexpression plasmid was also maintained in our laboratory. The GVRP1-ORF mouse polyclonal antibody was generated in our laboratory.

[0045] Example 1 IAV infection specifically induces lncRNA GVRP1 expression

[0046] 1. Cell culture:

[0047] The cells used in the present invention are DF1 cells, and the culture conditions are DMEM + 10% fetal bovine serum (FBS) + 1% penicillin-streptomycin double antibody (100 U / mL penicillin + 0.1 mg / mL streptomycin).

[0048] (1) Cell recovery: Preheat the water bath to 37°C and precool the centrifuge to 4°C; preheat complete culture medium (growth medium) and remove the cryovial from liquid nitrogen. After checking that there is no liquid nitrogen residue, quickly immerse the cryovial in a 37°C water bath and gently shake until completely thawed (≤ 2 minutes); transfer the cell suspension to a centrifuge tube and dilute it with 4-5 mL of preheated culture medium; centrifuge at 900-1000 rpm for 5 minutes and discard the supernatant.

[0049] (2) Inoculation and culture: Resuspend the cells in 1-2 mL of fresh culture medium and transfer them to a T25 culture flask; add culture medium to 6 mL and mix thoroughly by cross-mixing; place the cells in an incubator (37°C, 5% CO2, saturated humidity) and culture them for 48-72 hours; observe the cells for adhesion after 24 hours and change the medium for the first time after 48 hours.

[0050] (3) Cell passaging: When the cell confluence reaches 80%-90% (dense monolayer with clear edges), discard the old culture medium and gently rinse 1-2 times with pre-warmed PBS (to remove serum residues); add 1 mL of 0.25% trypsin (containing EDTA) and digest in a 37°C incubator for 2 minutes; add 2-3 mL of complete culture medium to terminate the digestion and gently pipette to form a single-cell suspension. Centrifuge the cell suspension (1000 rpm, 5 minutes) and discard the supernatant; resuspend the cells and aliquot into new culture flasks at a ratio of 1:2 to 1:4 (e.g., 2-4 T25 flasks); replenish the culture medium (6 mL for T25 flasks), mix well, and place in a 37°C incubator.

[0051] 2. Viral infection and sequencing:

[0052] (1) Virus infection: Resuscitate DF1 cells and inoculate them in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Incubate at 37°C and 5% CO2 incubator until the cells are 80%-90% confluent, and evenly plate the cells in a cell plate. Take the virus stock solution from the -80°C refrigerator and dilute it in a serum-free medium. Aspirate the PBS in the cells and add the diluted virus solution to each well to ensure that the cell layer is covered. Adsorb at 37°C for 1-2 hours, gently shaking the culture plate every 15-30 minutes to ensure that the virus is evenly exposed to the cells. After adsorption, aspirate the virus solution and wash twice with PBS to remove unadsorbed virus. Add maintenance medium containing 0.3% BSA, 1% double antibody and TPCK trypsin (0.35-1μg / mL). DF1 cells were infected with MOI = 0.01, 0.1, 1, 3, or MOI = 0.1, and total RNA was collected with trizol at different time points of 0, 6, 12, 24, and 36 h and stored in a -80°C laboratory ultra-low temperature freezer until use.

[0053] (2) Transcriptome sequencing: DF-1 cells were infected with H9N2 influenza A virus. After total RNA was extracted from the sample, ribosomal RNA was removed to maximize the retention of all coding RNA and ncRNA. The resulting RNA was randomly fragmented into short fragments. The fragmented RNA was then used as a template to synthesize the first-strand cDNA using random hexamers. Buffer, dNTPs (dUTP instead of dTTP), RNase H, and DNA polymerase I were then added to synthesize the second-strand cDNA. The cDNA was purified using a QiaQuick PCR kit and eluted with EB buffer. After end repair, base A addition, sequencing adapters were added, and the second-strand was degraded by UNG (Uracil-N-Glycosylase). Fragment size selection was performed by agarose gel electrophoresis, and PCR amplification was performed. The constructed sequencing library was sequenced using an Illumina HiSeqTM 4000. After filtering the offline data to obtain clean data, reads were aligned to the reference genome using Hisat2, and transcripts were reconstructed using StringTie to identify known and novel transcripts. The coding capacity of the novel transcripts was predicted using software such as CPC and CNCI, resulting in newly predicted lncRNAs. mRNA and lncRNA expression levels were then analyzed in each sample, followed by lncRNA-mRNA association analysis. Significant P values ​​were calculated using DESeq2 software, followed by multiple hypothesis testing correction. The P value threshold was then determined by controlling the FDR (False Discovery Rate) and corrected to obtain the corrected P value. Fold Change (FC) was also calculated to compare gene expression levels between different genes or experimental groups. Genes with differential expression between groups were screened based on log2(FC) >= 1 and P value <= 0.05. Furthermore, structural analysis of the resulting transcripts was performed, including gene structure optimization and alternative splicing analysis. Differentially expressed genes were clustered bidirectionally using the pearson correlation metric and the average linkage rule, respectively, and displayed as a heatmap. GO enrichment and functional analysis of the target genes was performed using TopGO software, primarily encompassing biological process (BP), cellular component (CC), and molecular function (MF). The KEGG database's pathway function was used to annotate and categorize the target genes, thereby identifying the signaling pathways in which the differentially expressed genes were primarily enriched.The protein interaction network of the species was queried and downloaded from the STRING protein interaction network database (http: / / string-db.org) to construct the protein interaction network of differentially expressed genes.

[0054] 3. RNA Extraction:

[0055] In an RNase-free environment, rinse the cells for RNA extraction three times with cold PBS. Add 1 mL of RNA trizol to each well and lyse on ice for 10 minutes. Pipette the cells repeatedly to thoroughly lyse and mix, then transfer to an RNase-free 1.5 mL EP tube and place on ice. Add 200 μL of pre-chilled chloroform to each sample, shake vigorously for 15 seconds until the emulsion becomes turbid, and let it stand on ice for 5 minutes. Centrifuge at 12,000 rpm at 4°C for 15 minutes. The liquid will separate into three layers. Transfer the upper, colorless aqueous phase to a new, RNase-free 1.5 mL EP tube. Add an equal volume of pre-chilled isopropanol to each tube, gently invert to mix, and let it stand at -20°C for 10-30 minutes. Centrifuge at 12,000 rpm at 4°C for 10 minutes. Carefully discard the supernatant and add 1 mL of pre-chilled 75% ethanol (prepared with DEPC water) to each sample. Invert several times to rinse the pellet and let it sit on ice for 3-5 minutes. Centrifuge at 12,000 rpm at 4°C for 5 minutes. Discard the supernatant completely and air-dry the pellet in a fume hood for 2-5 minutes. Add 20 μL of DEPC water to the pellet and gently pipette to mix thoroughly to dissolve it. This will yield RNA, which can be used directly for reverse transcription or stored at -80°C.

[0056] 4. cDNA Reverse Transcription:

[0057] The extracted RNA concentration was determined, and 1 μg of total RNA was reverse transcribed using the HiScript II 1st Strand cDNA Synthesis Kit (with gDNA wiper). Reverse transcription system: Total RNA ≤ 5 μg (volume variable), 2 μL of 4× gDNAwiper Mix, 8 μL of RNase-free ddH2O, gently pipetting to mix, and briefly centrifuge. Incubate at 42°C for 2 minutes in a PCR instrument. To this mix, add 4 μL of 5× HiScript II qRT SuperMix II, 2 μL of HiScript II Enzyme Mix, 1 μL of 50 μM Oligo(dT)23VN or Random Primer, and 5 μL of RNase-free ddH2O, and gently pipette to mix.

[0058] Reverse transcription protocol: Annealing: 25°C for 5 minutes; Extension: 50°C for 15 minutes. Enzyme inactivation: 85°C for 5 seconds. Storage: Use immediately or store at -20°C for short-term storage (-80°C aliquots are recommended for long-term storage).

[0059] 5. RT-qPCR:

[0060] Using cDNA as a template, perform quantitative PCR using the AceQ qPCR SYBR Green Master Mix Kit and gene-specific primers on a fluorescent quantitative PCR instrument. Quantitative PCR reaction components: 10.0 μL of 2× AceQ qPCR SYBR Green MasterMix, 0.4 μL of 50× ROX Reference Dye (optional), 0.4 μL of qPCR Primer F (10 μM), 0.4 μL of qPCR Primer R (10 μM), 2.0 μL of Template cDNA, and RNase-free ddH2O to a volume of 20 μL.

[0061] Cycling program: 95°C, 5 min; (95°C, 10 s; 60°C, 30 s) × 40 cycles; 95°C, 15 s; 60°C, 60 s; 95°C, 15 s.

[0062] The expression level of GAPDH was used as an internal reference, and the relative RNA level of the target gene was calculated using the 2^(-ΔΔCt) method.

[0063] qPCR-specific primer sequences:

[0064] lncRNA GVRP1 upstream qPCR primer (SEQ ID No .4): TCCGTTCTGGTGCTTGGC;

[0065] lncRNA GVRP1 downstream qPCR primer (SEQ ID No .5): CAACCGCTGCTTGCTCAC;

[0066] 6. Implementation Effect:

[0067] To analyze whether lncRNA can regulate influenza virus replication, we infected DF1 cells with influenza virus and performed whole transcriptome sequencing. We found that lncRNA molecules with significantly upregulated levels ( Figure 1), the nucleotide sequence is shown in SEQ ID No. 1. Sequencing analysis showed that the lncRNA sequence is 2633 nt long and is located on avian chromosome 7. To facilitate subsequent research, it was named lncRNA GVRP1. The secondary structure of lncRNA GVRP1 was depicted using the RNAfold website. Compared with before virus infection, lncRNA GVRP1 can induce its expression level to be significantly increased under infection with different subtypes of influenza virus ( Figure 2 Figure A). DF1 cells were infected with influenza virus at an MOI of 0.1, and RNA was extracted at 0h, 6h, 12h, 24h, and 36h after infection. The expression of lncRNA GVRP1 was detected by RT-qPCR. The results showed that the expression level of lncRNA GVRP1 increased with the increase of virus infection time ( Figure 2 Figure B). DF1 cells were infected with influenza virus at different MOIs (0, 0.01, 0.1, 1, and 3). 24 hours after infection, cells were harvested, RNA was extracted, and the expression of lncRNA GVRP1 was detected by RT-qPCR. The results showed that the expression level of lncRNA GVRP1 increased with the increase of viral infection dose ( Figure 2 These results indicate that lncRNA GVRP1 is specifically upregulated by influenza virus in an infection time- and virus dose-dependent manner.

[0068] Example 2 lncRNA GVRP1 is involved in regulating influenza virus replication

[0069] 1. Construction of lncRNA GVRP1 overexpression plasmid:

[0070] (1) Amplification of target fragment: The single-stranded cDNA obtained by reverse transcription is used to amplify the full-length DNA fragment of lncRNA GVRP1 through PCR reaction using specific primers and procedures and high-fidelity enzymes.

[0071] PCR specific primer sequences:

[0072] lncRNA GVRP1 upstream primer (SEQ ID No .6): tagtccagtgtggtggaattcCAGGTAAGCTTCCTGATGGTGTCA;

[0073] lncRNA GVRP1 downstream primer (SEQ ID No. 7): aacgggccctctagactcgagCGTTTCTCAGGAGGAGC.

[0074] (2) Recovery of target fragment and vector digestion: The PCR product was subjected to nucleic acid electrophoresis. DNA was purified and recovered using a gel recovery kit. The pcDNA3.1 plasmid was digested with EcoRI and XhoI. The reaction system was as follows: 5 μL of 10× Buffer, 2000 ng of plasmid, 1 μL each of EcoRI and XhoI, and ddH2O was added to 50 μL. The reaction conditions were 37°C for 6 h and inactivated at 80°C for 20 min. The fragment was recovered by agarose gel electrophoresis (same steps as above) and set aside.

[0075] (3) Ligation of the target fragment and vector: Use the ClonExpress II One Step Cloning Kit to ligate the target fragment and the enzyme-digested vector. Reaction system: 0.03 pmol of cloning vector, 0.06 pmol of insert, 4 μL of 5 × CE II Buffer, 2 μL of Exnase II, and ddH2O to 20 μL. Use a pipette to gently pipette to mix, briefly centrifuge to collect the reaction solution at the bottom of the tube, and react at 37°C for 30 min; then cool to 4°C or immediately place on ice.

[0076] (4) Transformation of recombinant products and plasmid extraction: Thaw cloning competent cells (DH5α Competent cells) on ice. Take 10 μl of recombinant product and add it to 100 μl of competent cells, gently tap the tube wall to mix (do not oscillate to mix), and let it stand on ice for 30 minutes. After heat shock in a 42°C water bath for 45 seconds, immediately place it on ice to cool for 2-3 minutes. Add 900 μl of LB medium (without antibiotics) and shake the culture at 37°C for 1 hour (speed 200-250 rpm). Centrifuge at 5,000 rpm (2,400 × g) for 5 minutes and discard 900 μl of supernatant. Resuspend the bacteria in the remaining culture medium and gently spread it evenly on the plate containing the correct resistance with a sterile spreader. Incubate inverted at 37°C incubator for 12-16 hours. Pick the colony for positive identification and transfer the positive colony to 10 mL of culture medium for expansion culture. Use a plasmid extraction kit to extract the plasmid from the positive bacterial solution.

[0077] 2. Overexpression plasmid and siRNA cell transfection and infection:

[0078] (1) Exogenous overexpression of lncRNA GVRP1: Select DF1 cells in the logarithmic growth phase and in good condition, and seed them into 24-well plates one day before transfection, so that the cell confluence reaches 70%-90% at the time of transfection. Next, prepare the plasmid lncRNA GVRP1-pcDNA3.1 and the transfection reagent Lipofectamine 2000, dilute them with serum-free medium, mix them, and let them stand at room temperature for 20 minutes to form a transfection complex. Then, aspirate the cell culture medium, add serum-free medium, and then add the transfection complex dropwise to the cells and culture at 37°C for 6-8 hours. Finally, replace the complete medium, continue to culture for 24 hours, and then detect the expression level of the target gene by qPCR.

[0079] (2) Exogenous knockdown of lncRNA GVRP1: The cells were seeded into 24-well culture plates one day before transfection, so that the cell density reached 60%-80% at the time of transfection. On the day of transfection, siRNA (final concentration 20-100nM) and transfection reagent (Lipofectamine 2000) were diluted in serum-free medium (such as Opti-MEM), mixed in proportion, and allowed to stand at room temperature for 15-20 minutes to form a transfection complex. The original cell culture medium was aspirated and replaced with serum-free medium. The transfection complex was then added dropwise to the cells and gently shaken to mix. After 4 hours of transfection, the cells were replaced with complete medium (containing serum) and cultured for another 24 hours. The gene silencing effect was detected by qPCR. A negative control (NC) was required to avoid RNase contamination.

[0080] siRNA sequence:

[0081] lncRNA GVRP1-si1 (SEQ ID No.8): sense GACCGAGGGACAUUUAAUATT;

[0082] lncRNA GVRP1-si2 (SEQ ID No.9): sense GCAGGUGUCAUGCAAAUGUTT.

[0083] (3) Referring to step 2 of Example 1, the transfected cells were infected with the virus.

[0084] 3. Plaque assay:

[0085] MDCK cells were seeded into 12-well plates (3 × 10 5Cells were cultured at 37°C until a monolayer formed. The collected infection supernatant was then serially diluted 10-fold in serum-free DMEM. The cell culture medium was discarded, and the diluted virus solution was added for adsorption at 37°C for 1 hour (with gentle shaking every 15 minutes). After adsorption, the virus solution was discarded, the cells were washed with PBS, and DMEM medium containing 2% agarose and TPCK-trypsin was added. After solidification, the plate was inverted and incubated at 34°C for 48 hours. The cells were fixed with 4% paraformaldehyde, the agar layer was removed, and the cells were stained with crystal violet. Plaques were counted and the titer was calculated.

[0086] 4. Implementation Effect

[0087] To analyze whether lncRNA GVRP1 plays a regulatory role in influenza virus replication, we constructed an overexpression plasmid for lncRNA GVRP1 and designed and synthesized a specific siRNA for lncRNA GVRP1 gene silencing. The expression level of lncRNA GVRP1 was detected by qPCR, which proved that lncRNA GVRP1 was successfully overexpressed in DF1 cells ( Figure 3 Figure A in the figure) and silencing lncRNA GVRP1 ( Figure 3 Overexpression of lncRNA GVRP1 significantly promoted the proliferation of H9N2 influenza virus in DF1 cells, and the virus titer increased by about 4 times compared with the empty vector control group ( Figure 3 The same results were observed in the replication of different subtypes of influenza viruses, such as H1N1 ( Figure 3 C in Figure ) and H3N2 ( Figure 3 The designed specific siRNA was transfected into DF1 cells, which significantly inhibited the replication of H9N2 subtype influenza virus. Compared with the control group NC, the virus titer was reduced by about 2 times ( Figure 3 (Figure F in Figure ). The results demonstrated that lncRNA GVRP1 is a positive regulator involved in regulating influenza virus replication.

[0088] Example 3 lncRNA GVRP1 encodes a polypeptide with 75 amino acids

[0089] 1. Western Blot:

[0090] Proteins were extracted from transfected or infected cells, lysed using NP-40 lysis buffer (containing protease inhibitors), and the supernatant was centrifuged and the protein concentration was determined by the BCA method. SDS-PAGE electrophoresis was then performed, and the protein sample was mixed with loading buffer, denatured by boiling, and loaded onto a stacking gel (80V) and a separation gel (120V). After electrophoresis, the protein was transferred to a NC membrane using a semi-dry transfer method (200mA, 30 minutes), and then blocked with 5% skim milk powder or BSA at room temperature for 1 hour to block nonspecific binding. The membrane was then incubated with the primary antibody (overnight at 4°C) and the HRP-labeled secondary antibody (1 hour at room temperature), followed by washing three times with TBST after each incubation. The membrane was finally developed using an ECL chemiluminescent substrate, and the signal was detected using an imaging system to analyze the expression level of the target protein (correction with an internal reference protein such as GAPDH was required).

[0091] 2. Construction of GVRP1-ORF tag protein and fluorescent protein:

[0092] The full-length sequence was amplified using GVRP1-ORF-specific primers and constructed on C-myc-pcDNA3.1 and GFP-pcDNA3.1 vectors, respectively. The restriction sites were EcoRI and XhoI, and the specific operation steps were the same as those in Step 1 of Example 2. Colonies were picked for positive identification, and the positive colonies were transferred to 10 ml of culture medium for expansion culture. Plasmids were extracted from the positive bacterial solution.

[0093] The GFP-pcDNA3.1 overexpression plasmid signal peptide region was double-mutated using a point mutation method to construct a GPF-mut-pcDNA3.1 mutant plasmid. Subsequently, the sequence of GVRP1-ORF was constructed in the GPFmut-pcDNA3.1 vector, the target sequence was inserted into the front end of the GFP-mut sequence, and the fragment was connected to the vector by homologous recombination to construct a plasmid named GVRP1-ORF-GFPmut-pcDNA3.1. Colonies were picked for positive identification, and the positive colonies were transferred to 10 ml of culture medium for expansion culture, and plasmid extraction was performed on the positive bacterial liquid. In the same steps, the start codon of the GVRP1-ORF sequence was double-mutated and constructed on the GPFmut-pcDNA3.1 vector. The plasmid name was ORFmut-GFPmut-pcDNA3.1.

[0094] 3. Cellular localization of GVRP1-ORF protein:

[0095] When DF1 cells reached 80-90% confluency in a 12-well plate, 1 μg of GFP-pcDNA3.1, 1 μg of GFPmut-pcDNA3.1, and 1 μg of GVRP1-ORF-GFPmut-pcDNA3.1 were transfected into the cells according to the Lip2000 instructions. After 36 hours of culture, the cells were observed for fluorescence using an inverted fluorescence microscope.

[0096] When DF1 cells reached 60-70% confluency in a confocal microplate, 2 μg of GVRP1-ORF-GFPmut-pcDNA3.1 was transfected into the cells according to the Lip2000 instructions. After 48 hours of culture, the distribution of green fluorescent protein in the cells was observed using a confocal microscope.

[0097] 4. Implementation effect:

[0098] Through combined RNA-seq and RIBO-seq analysis, combined with predictions from bioinformatics websites such as ORF finder, we conducted preliminary verification of potential long non-coding RNAs with coding capabilities and found that lncRNA GVRP1 has the potential to encode an ORF. Analysis showed that GVRP1-ORF is a polypeptide sequence containing 75 amino acids (sequence shown in SEQ ID NO. 3). Alphafold3 software was used to simulate the GVRP1-ORF protein structure, and the results are as follows: Figure 4 As shown in Figure C. The constructed GVRP1-ORF-myc-pcDNA3.1 and control myc-pcDNA3.1 plasmids were then transfected into DF1 cells, and the cell lysates were collected for western blot. The results showed that compared with the empty control group, the GVRP1-ORF-myc-pcDNA3.1 overexpression sample had a clear protein band at 15kDa ( Figure 4 Figure A in the figure demonstrates that GVRP1-ORF has translational potential. Furthermore, by constructing ORF-GFP and its mutant plasmids and testing the expression of ORF-GFP fusion protein, the results showed that DF1 cells transfected with GFP-pcDNA3.1 and ORF-GFPmut-pcDNA3.1 plasmids could clearly express GFP ( Figure 4(B in Figure 1), while no green fluorescent protein expression was observed in DF1 cells transfected with GFPmu-pcDNA3.1 and ORFmut-GFPmut-pcDNA3.1 plasmids. This further verified that the GVRP1-ORF signal peptide has translational activity and the ability to encode polypeptides. Fluorescence co-localization of proteins overexpressing ORF-GFPmut-pcDNA3.1 showed that the GVRP1-ORF protein was mainly localized in the cytoplasm ( Figure 4 Figure D in the figure).

[0099] Example 4 GVRP1-ORF overexpressing cell line has the ability to significantly enhance influenza virus replication

[0100] 1. Construction of GVRP1-ORF overexpressing cell line:

[0101] (1) Construction of PLOX-GVRP1-ORF-IRES-TK plasmid: The specific operation steps are the same as step 1 of Example 2. First, the target gene fragment is amplified from the target template by PCR; at the same time, the PLOX-IRES-TK vector is double-digested with the same enzyme, the purified target fragment and the vector are mixed at a molar ratio of 1:2-3, homologous recombinase is added for homologous recombination, the ligation product is transformed into DH5α competent cells, and LB plates containing corresponding antibiotics (such as ampicillin) are coated. Single colonies are picked for colony PCR and sequencing verification, and the correct recombinant plasmid is extracted for use.

[0102] (2) Cell transfection and screening: The specific operation steps are the same as those in step 2 of Example 2. The successfully verified recombinant plasmid is transfected into the target cells (DF1 cells) by liposome method (Lip2000). After 48 hours of transfection, the screening antibiotic (G418, concentration is 500 μg / mL) is added, and the screening is continued for 7-14 days. The medium is changed every 2-3 days to maintain the pressure. Untransfected cells are set as negative controls and PLOX-EGFP-IRES-TK fluorescent plasmid is set as positive controls for screening. After the screening is completed, the endogenous expression of the protein is verified by Western blot. The positive cell lines are expanded and frozen for future use.

[0103] 2. Cell viability detection of stably overexpressing cell lines:

[0104] Collect overexpression cell lines and control cells (such as empty vector transfected cells) in the logarithmic growth phase, trypsinize and centrifuge (1,000 rpm, 5 min), and resuspend in serum-containing medium. Adjust the cell density to 5×10³~5×10 4Seed cells at 100 μL / mL into a 96-well plate, with 100 μL of cell suspension per well. Pre-incubate the plate in a 37°C, 5% CO2 incubator for 24 hours to allow the cells to attach and reach a stable state. If drug treatment is desired, add the test substance at varying concentrations at this step and continue incubation for 6 to 96 hours. Add 10 μL of CCK-8 solution directly to each well (avoiding air bubbles) and gently shake the plate to mix. Continue incubation for 1 to 4 hours (37°C, 5% CO2). Measure the OD value of each well at 450 nm using a microplate reader.

[0105] 3. Implementation effect:

[0106] By constructing the PLOX-GVRP1-ORF-IRES-TK plasmid, the positive plasmid was transferred into DF1 cells to screen the positive cells stably expressing the GVRP1-ORF protein. The number of green fluorescent cells in the positive control well PLOX-EGFP-IRES-TK showed the effect of screening positive cells ( Figure 5 As shown in Figure A), a portion of the cell lysate was collected from the positive wells screened to detect the endogenous expression of GVRP1-ORF protein in the cells. The results showed that compared with wild-type cells, the six selected positive cell lines were able to stably express GVRP1-ORF protein with higher expression abundance ( Figure 5 At the same time, CCK8 results showed that there was no significant difference in cell viability between the stably transfected cell lines and wild-type cells ( Figure 5 (Figure C in the figure) The results demonstrated the successful construction of a stably transfected cell line overexpressing the GVRP1-ORF protein. Subsequently, wild-type DF1 cells and the overexpressing cell line were infected with H9N2 virus at an MOI of 1. Cell supernatants, cellular RNA, and cell protein lysates were collected 24 hours after infection. Plaque titration of influenza virus titers revealed that cells stably overexpressing the GVRP1-ORF protein promoted influenza virus proliferation, with viral titers approximately threefold higher than those of wild-type cells ( Figure 5 Similarly, the mRNA and protein levels of influenza virus NP gene were detected by fluorescent quantitative PCR and western blot, showing the same results ( Figure 5 These results indicate that the construction of a GVRP1-ORF protein stably transfected cell line can significantly enhance influenza replication, providing new insights into the application of candidate cell lines in subsequent influenza vaccine production.

[0107] Example 5 GVRP1-ORF is the main functional domain of lncRNA GVRP1 involved in regulating influenza virus replication

[0108] 1. Construction of plasmids encoding different truncated lncRNA GVRP1

[0109] lncRNA GVRP1 was truncated to construct different truncated overexpression plasmids: lncRNA GVRP1 (1-2633 bp, full length), GVRP1-ORF (82-306 bp, expressed ORF region), lncGVRP1-ΔORF (1-81&207-2633, deleted ORF region), lncGVRP1-A (1-1344), and lncGVRP1-B (1345-2633). The specific construction steps were the same as those in step 1 of Example 2.

[0110] 2. Implementation effect:

[0111] To further explore the relationship between lncRNA GVRP1 and its encoded polypeptide in regulating influenza virus replication, lncRNA GVRP1 was truncated to find the functional domain of lncRNA GVRP1 that plays a specific regulatory role. The truncation diagram is shown below. Figure 6 As shown in Figure A. The results showed that overexpression of lncRNA GVRP1 and its polypeptide can promote influenza virus proliferation. Overexpression of lncRNA GVRP1-A segment (the first half of the sequence) also has a regulatory effect. However, overexpression of only lncRNA GVRP1-B segment (the second half of the sequence) or deletion of GVRP1-ORF segment did not affect viral replication compared with the control group ( Figure 6 Therefore, the results indicate that GVRP1-ORF is the main functional domain of lncRNA GVRP1 involved in regulating influenza virus replication.

[0112] To further analyze the possible mechanism of action of lncRNA GVRP1 in influenza virus replication, we performed co-expression analysis of lncRNA GVRP1 and differential mRNA based on the whole transcriptome sequencing results of previous influenza infection of host cells. We used the WGCNA method to cluster genes with similar expression patterns to obtain different co-expression modules, and performed KEGG-Pathway functional enrichment analysis on the protein-coding genes within the modules to predict the main functions of lncRNAs within the modules. The results showed that genes co-expressed with lncRNA-DARVR were mainly clustered in Lysosome and ABC transporters ( Figure 7During the early stages of influenza virus replication, the virus binds to sialic acid receptors (α2,3 / α2,6 linkage) on the host cell surface via hemagglutinin (HA), triggering clathrin-mediated endocytosis and the formation of early endosomes. Following endosome acidification, HA conformational changes expose the fusion peptide, mediating fusion of the viral envelope with the endosomal membrane and releasing the viral ribonucleoprotein (vRNP) into the cytoplasm. Gradual endosome acidification (pH ≈ 5.0) is a key step in HA-activated fusion, which is dependent on lysosomal-associated membrane proteins (LAMPs) and proton pumps (such as v-ATPase). ABC transporters (such as TAP1 / TAP2) participate in the endoplasmic reticulum transport of antigenic peptides to MHC class I molecules, influencing viral antigen presentation and CD8⁺ T cell responses. Therefore, the above pathways suggest that lncRNA GVRP1 may be involved in influenza virus endocytosis and membrane fusion. For example, ABC transporters may help maintain the Ca²⁺ / H⁺ gradient within the lysosomal lumen, influencing M2 ion channel activity and indirectly regulating endosomal fusion efficiency.

[0113] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the appended claims.

Claims

1. lncRNA, characterized in that The nucleotide sequence thereof is shown in SEQ ID No. 1; the RNA sequence transcribed therefrom is shown in SEQ ID No.

2.

2. The polypeptide encoded by the lncRNA according to claim 1, characterized in that Its amino acid sequence is shown in SEQ ID NO.

3.

3. A vector containing the lncRNA according to claim 1.

4. Use of the lncRNA according to claim 1, the polypeptide according to claim 2, or the vector according to claim 3 in screening candidate substances for preventing and treating influenza virus, wherein the influenza virus is influenza virus H9N2.

5. The lncRNA inhibitor according to claim 1, characterized in that The inhibitor is si-lncRNA, and its sequence is any one of SEQ ID NOs: 8 to 9.

6. A pharmaceutical composition for preventing and treating influenza virus, characterized in that: The pharmaceutical composition contains the inhibitor according to claim 5, and the influenza virus is influenza virus H9N2.

7. Use of the inhibitor according to claim 5 or the pharmaceutical composition according to claim 6 in the preparation of a medicament for preventing and treating avian influenza virus, wherein the influenza virus is influenza virus H9N2.

8. The use according to claim 7, characterized in that The drug further includes a pharmaceutically acceptable carrier.

9. A method for inhibiting influenza virus proliferation in vitro, characterized in that: The method comprises administering the inhibitor of claim 5, wherein the influenza virus is influenza virus H9N2, and the in vitro expression is in DF1 cells.

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