FLNB gene-knockout MDCK cell line, construction method therefor, and use thereof

Abstract

Provided are an FLNB gene-knockout MDCK cell line, a construction method therefor and the use thereof. The FLNB gene-knockout cell line is named MDCK-FLNB-KO, and has been deposited in China Center for Type Culture Collection (CCTCC) on November 20, 2024, and the accession number is CCTCC NO: C2024391. The FLNB gene knockout uses CRISPR / Cas9 technology, wherein a sgRNA is designed for exon 1 of the FLNB gene of MDCK cells, an FLNB gene knockout vector plasmid 1 and plasmid 2 are constructed, and the plasmid 1 and the plasmid 2 are transfected into the MDCK cells to obtain the FLNB gene-knockout MDCK cell line. Compared with the original MDCK cell line, the obtained cell line has a lower adhesion ability and a higher yield when applied to influenza virus culture.

Description

FLNB gene knockout MDCK cell line, its construction method and application Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an FLNB gene knockout MDCK cell line, its construction method, and its application. Background Technology

[0002] MDCK (Madin-Darby Canine Kidney cells) are a cell line isolated from canine kidney tissue. MDCK cells grow rapidly, are highly adaptable, easy to culture, and can survive under various culture conditions. Due to the presence of numerous influenza virus-sensitive receptors on their cell surface, MDCK cells are an ideal cell line for culturing influenza viruses. However, native MDCK cells exhibit strong adhesion, high clumping rate, and long digestion time, which are unfavorable for cell culture and large-scale production. Domesticated suspension cells, on the other hand, have higher passage counts, increased tumorigenicity, unclear mechanisms, and the risk of reversible domestication, making them unsuitable for use in influenza vaccine manufacturing. FLNB (Filamin B) expresses filamentin B, an actin-binding protein mainly located in the cytoplasm. Its structure consists of two homologous subunits non-covalently bound. FLNB participates in the construction, maintenance, and dynamic changes of the cytoskeleton through interactions with other cytoskeletal proteins and can also mediate signal transduction with adhesion molecules, which is crucial for cell morphology, adhesion, and migration. Therefore, this study used CRISPR-Cas9 gene editing technology to construct a low-adhesion MDCK cell line with FLNB gene knockout, which is of great significance for promoting the better application of MDCK cells in the manufacture of influenza vaccines. Summary of the Invention

[0003] In view of this, the present invention provides an MDCK cell line with FLNB gene knockout, its construction method and application, by using CRISPR-Cas9 gene editing technology to knock out the FLNB gene in MDCK cells to obtain an MDCK cell line with reduced adhesion ability.

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

[0005] In a first aspect, this invention provides a method for FLNB gene knockout. Using CRISPR / Cas9 technology, sgRNA is designed targeting exon 1 of the FLNB gene in MDCK cells to construct FLNB gene knockout vector plasmids 1 and 2. Plasmids 1 and 2 are transfected into MDCK cells to obtain a FLNB gene knockout MDCK cell line. The FLNB gene knockout cell line is named MDCK-FLNB-KO and was deposited at the China Center for Type Culture Collection (CCTCC) on November 20, 2024, with accession number CCTCC NO: C2024391.

[0006] Preferably, the nucleotide sequence of the sgRNA is shown in SEQ ID No. 1.

[0007] Preferably, plasmid 1 is pX330-sgRNA plasmid.

[0008] Preferably, plasmid 2 is pY75-5-3 plasmid.

[0009] Preferably, the pY75-5-3 plasmid contains a 5' homologous arm and a 3' homologous arm for the sequence recognition site of the sgRNA, the sequence of the 5' homologous arm is shown in SEQ ID No. 2, and the sequence of the 3' homologous arm is shown in SEQ ID No. 3.

[0010] Secondly, the present invention provides a FLNB gene knockout MDCK cell line, wherein the FLNB gene knockout MDCK cell line is obtained according to the method described above.

[0011] Finally, the present invention provides an application of the method for obtaining the FLNB gene knockout MDCK cell line, or the FLNB gene knockout MDCK cell line, in the preparation of an influenza virus vaccine.

[0012] Preferably, the influenza virus includes influenza A virus and / or influenza B virus.

[0013] Preferably, the influenza virus includes at least one of the subtypes of influenza viruses: H1N1, H3N2, BV, and BY.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] (1) This invention uses CRISPR-Cas9 gene editing technology to knock out the FLNB gene in MDCK cells to obtain MDCK cells with reduced adhesion ability.

[0016] (2) The FLNB gene knockout MDCK cell line provided by this invention has similar cell proliferation ability to the original MDCK cell line, but the total number of cells in the plateau phase is slightly larger.

[0017] (3) Compared with the original MDCK cell line, the FLNB gene knockout MDCK cell line provided by the present invention grows loosely and has a lower clumping rate, which is conducive to cell digestion.

[0018] (4) Compared with the original MDCK cell line, MDCK cells with FLNB gene knockout showed improved cell-matrix adhesion and increased intracellular calcium content. 2+ The concentrations all decreased. Trypsin digestion at 95% cell confluence significantly reduced the digestion time of FLNB gene knockout MDCK cells, substantially improving cell passage efficiency and reducing cell digestion time, which is beneficial for large-scale influenza vaccine production.

[0019] (5) The expression of the FLNB gene may negatively regulate the entry of the virus into the host cell. MDCK cells with FLNB gene knockout can increase the hemagglutination titer of various influenza subtypes, and therefore can be used for the preparation of influenza virus vaccines. Attached Figure Description

[0020] Figure 1 is a spectral image of the pX330 plasmid provided in Example 1 of the present invention;

[0021] Figure 2 is a gel electrophoresis diagram of the pX330-sgRNA plasmid provided in Example 1 of the present invention;

[0022] Figure 3 is a spectrum of the pY75 plasmid provided in Example 1 of the present invention;

[0023] Figure 4 is a gel electrophoresis diagram of the pY75-5-3 plasmid provided in Example 1 of the present invention;

[0024] Figure 5 shows the gel electrophoresis diagrams of the original MDCK cell line and the FLNB gene knockout MDCK cell line provided in Example 1 of the present invention.

[0025] Figure 6 shows the qPCR results of mRNA expression levels in the original MDCK cell line and the FLNB gene knockout MDCK cell line provided in Example 2 of this invention.

[0026] Figure 7 shows the protein expression level analysis of the original MDCK cell line and the FLNB gene knockout MDCK cell line provided in Example 2 of the present invention.

[0027] Figure 8 shows the growth curves of the original MDCK cell line and the FLNB gene knockout MDCK cell line provided in Example 3 of the present invention.

[0028] Figure 9 is an observation diagram of plate cell clone formation of the original MDCK cell line and the FLNB gene knockout MDCK cell line provided in Example 3 of the present invention.

[0029] Figure 10 shows the cell passage growth characteristics of the original MDCK cell line and the FLNB gene knockout MDCK cell line provided in Example 3 of the present invention.

[0030] Figure 11 shows the cellular fluorescence staining of the original MDCK cell line and the FLNB gene knockout MDCK cell line provided in Example 4 of the present invention.

[0031] Figure 12 is a comparison of cell adhesion strength between the original MDCK cell line and the FLNB gene knockout MDCK cell line provided in Example 4 of the present invention.

[0032] Figure 13 shows the calcium ion concentration detection of the original MDCK cell line and the FLNB gene knockout MDCK cell line provided in Example 4 of the present invention.

[0033] Figure 14 is a comparison of trypsin digestion between the original MDCK cell line and the FLNB gene knockout MDCK cell line provided in Example 4 of the present invention.

[0034] Figure 15 is a comparison of cell scratch assays between the original MDCK cell line and the FLNB gene knockout MDCK cell line provided in Example 5 of the present invention.

[0035] Figure 16 shows the soft agar clone formation of the original MDCK cell line and the FLNB gene knockout MDCK cell line provided in Example 5 of the present invention.

[0036] Figure 17 is a comparison of the susceptibility of the original MDCK cell line and the FLNB gene knockout MDCK cell line provided in Example 6 of the present invention to various subtypes of influenza virus. Detailed Implementation

[0037] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0038] The sources or components of the reagents, kits, or other materials used in the following examples are as follows:

[0039] MDCK cell line: purchased from ATCC, catalog number CCL-34, W-63 (working bank).

[0040] VP-SFM medium (catalog number: 12559019), fetal bovine serum (catalog number: 16136071), and glutamine additive (catalog number: 31980030) were all purchased from GIBCO.

[0041] The pX330 plasmid carries an sgRNA backbone, a Cas9 expression sequence, and an anti-ampicillin gene. The pY75 plasmid carries a loxP fragment containing the puro resistance gene.

[0042] 2×Phanta Max Master Mix (Dye Plus) (item number: P525), 2×Phanta Flash Master Mix (Dye Plus) (item number: P520), and T4 PNK (item number: N102) were all purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0043] T7DNA Ligase (Catalog No.: M0318S) was purchased from NEB.

[0044] The top 10 competent cells were purchased from Sangon Biotech (Shanghai) Co., Ltd. (item number: B528412).

[0045] The following influenza virus strains were stored at Wuhan Institute of Biological Products Co., Ltd. in China: H1N1 (IVR215) strain with batch number 202210W05H1, H3N2 (IVR237) strain with batch number 202401W01H3, BV (BVR-26) strain with batch number 202303W01BV, and BY (BVR-1B) strain with batch number 202205W02BY.

[0046] The methods not described in detail in the following embodiments are all conventional methods well known to those skilled in the art.

[0047] Example 1

[0048] This embodiment provides a method for constructing an MDCK cell line with FLNB gene knockout, including the following steps:

[0049] 1. Design of sgRNA for the FLNB gene in MDCK cells

[0050] The sgRNA sequence targeting exon 1 of the FLNB gene (NCBI website Gene ID: 484713) in MDCK cells was designed using the sgRNA design website http: / / www.e-crisp.org / E-CRISP /

[0051] SEQ ID No.1: 5'-GGTAACCGAGAAGGACCTGG-3'

[0052] And synthesize single-chain F: 5'-CACCGGTAACCGAGAAGGACCTGG-3'

[0053] R: 5'-AAACCCAGGTCCTTCTCGGTTACC-3'

[0054] Take 1 μL of each of the two single chains, 1 μL of T4 Ligation Buffer, 6.5 μL of ddH2O, and 0.5 μL of T4 PNK, for a total of 10 μL. Perform phosphorylation and annealing reactions at 37 °C for 30 min; 95 °C for 5 min; and decrease the temperature by 5 °C per minute until reaching 25 °C.

[0055] 2. Constructing the pX330-sgRNA plasmid

[0056] The pX330 plasmid (plasmid map shown in Figure 1) was ligated to the synthesized double-stranded sgRNA using a one-step enzyme digestion and ligation method to obtain the pX330-sgRNA plasmid. The reaction system consisted of: 100 ng pX330 plasmid, 2 μL double-stranded sgRNA reaction dilution buffer, 2 μL Tango buffer, 1 μL DTT, 1 μL ATP, 1 μL BBSI enzyme, 0.5 μL T7 DNA ligase, and ddH2O to a total reaction volume of 20 μL. The reaction program was: 37℃ for 30 min; 23℃ for 30 min.

[0057] Transform competent cells with the reaction product. Gently mix the reaction solution into E. coli TOP10 competent cells, incubate on ice for 30 min, heat shock at 42°C for 45 s, immediately return to ice for 2 min, add 700 μL of antibiotic-free sterile medium, and incubate at 37°C with shaking for 1 h (160-225 rpm). Spread an appropriate volume evenly onto LB agar plates containing the appropriate antibiotic and incubate overnight. Single colonies are picked for colony PCR identification using the following primers:

[0058] Upstream primer: 5'-CACCGGTAACCGAGAAGGACCTGG-3'

[0059] Downstream primer: 5'-CTTGATGTACTGCCAAGTGG-3'

[0060] The reaction system consisted of 2 μL bacterial culture, 1 μL upstream primer, 1 μL downstream primer, 12.5 μL 2×PhantaMax Master Mix, and 8.5 μL ddH2O. The reaction program was as follows: pre-denaturation at 98℃ for 30 s; denaturation at 95℃ for 10 s; annealing at Tm temperature for 5 s; extension at 72℃ for 5 s; amplification for 30 cycles; and final extension at 72℃ for 1 min. The reaction products were verified by 1% agarose gel electrophoresis, and the results are shown in Figure 2. Colonies with a band size of approximately 500 bp were considered positive. These positive single colonies were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0061] 3. Design of homologous arms flanking sgRNA

[0062] Homologous arm sequences were designed by selecting sequences approximately 1000 bp in length upstream and downstream of the sgRNA sequence:

[0063] 5' end homologous arm SEQ ID No. 2:

[0064] 3' Homologous Arm SEQ ID No. 3:

[0065] Homologous arm sequences were obtained using wild-type MDCK cell genomic DNA as a template via PCR amplification.

[0066] The primers for PCR amplification of the 5' homologous arm are as follows:

[0067] Upstream primer: 5'-CCATCGATCCATGAAGGGAGCCCGAG-3'

[0068] Downstream primer: 5'-GGGATATCCGAACTCGCTGCTACTGG-3'

[0069] The primers for PCR amplification of the 3' homologous arm are as follows:

[0070] Upstream primer: 5'-ACGCGTCGACCAAGCGCATCGGGAACCT-3'

[0071] Downstream primer: 5'-CCCAAGCTTCCTTCCCACTTCCATGACC-3'

[0072] The reaction mixture consisted of MDCK-WT DNA (100 ng), 2 μL upstream primer, 2 μL downstream primer, 25 μL 2×Phanta Max Master Mix, and ddH2O to a total volume of 50 μL. The reaction program was as follows: pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 15 s, annealing at Tm temperature for 15 s, extension at 72 °C for 60 s, for 35 cycles; and final extension at 72 °C for 5 min.

[0073] 4. Construct the homologous recombination vector pY75-5-3 plasmid

[0074] The 5' homologous arm and the pY75 plasmid (plasmid map shown in Figure 3) were digested with ClaI and EcoRIV enzyme (NEB) and incubated overnight at 37°C. The digestion products were purified using a Universal DNA Purification Kit (catalog number: DP214, TIANGEN). The purified products were ligated using the NEB T4 DNA Ligase system and incubated overnight at 16°C. The ligation products were transformed into competent cells, and single colonies were picked for PCR and gel electrophoresis identification. The corresponding positive single colonies were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results showed that the 5' homologous arm was correctly ligated. The 3' homologous arm was ligated using the same method, with SalI and HindIII enzymes (NEB) used for the 3' homologous arm, finally yielding the homologous recombinant pY75-5-3 plasmid. The results are shown in Figure 4, where the colonies with a band size of approximately 1200 bp are positive colonies.

[0075] The primers for PCR identification of the 5' homologous arm are as follows:

[0076] Upstream primer: 5'-GCTGCAAGGCGATTAAGTTG-3'

[0077] Downstream primer: 5'-CATGCTCCAGACTGCCTTG-3'

[0078] The primers for PCR identification of the 3' homologous arm are as follows:

[0079] Upstream primer: 5'-GTGGTTTGTCCAAACTCATC-3'

[0080] Downstream primer: 5'-TGTGTGGAATTGTGAGCG-3'.

[0081] 5. Cell electroporation and screening of monoclonal cells

[0082] The successfully constructed pX330-sgRNA plasmid and pY75-5-3 plasmid were simultaneously electroporated into MDCK cells using an electroporator (Gene PulserXcell, Bio-Rad). The electroporation system contained 10-1 cells per electroporation cup. 6 8 μg each of pX330-sgRNA plasmid and pY75-5-3 plasmid were added to a total volume of 200 μL with electroporation buffer. Electroporation conditions were: pulse voltage 110 V; number of pulses 1; pulse width 25 ms. After electroporation, cells from each electroporation cuvette were aliquoted into 6-well cell culture plates, and complete cell culture medium was added. The plates were then incubated at 37°C.

[0083] Based on cell growth status, when cell confluence reaches 60%, the medium is replaced with complete medium containing 8 μg / mL puromycin for selection. The puromycin selection medium is changed every 2 days. After about one week, small cell clusters with puromycin resistance can be observed. Monoclonal cells are obtained through extreme dilution: First, the cell clusters with puromycin resistance that have grown in the six-well plates are digested; second, the cells are counted and diluted to a cell concentration of 30-50 cells / 10mL; finally, the cell suspension is thoroughly mixed and added to 100 μL per well of a 96-well plate, and cultured in a cell culture incubator at 37°C. Cell growth status is closely observed, and wells containing only monoclonal cell clusters are marked under a light microscope; after the cells in the marked wells have reached confluence, cell expansion culture is performed for cell identification and cell cryopreservation.

[0084] 6. Monoclonal cell PCR identification

[0085] Primers were designed upstream and downstream of the homologous arms at both ends of the inserted genomic sequence for PCR identification. This primer pair can simultaneously identify wild-type, single knockout, and double knockout clones, and eliminate interference from plasmid residues in the cell. First, genomic DNA was extracted from single-clonal cells using the Minibest universal Genomic DNA Extraction Kit (TAKARA) as a template for PCR identification. The reaction system consisted of: DNA template (100 ng), 1 μL upstream primer, 1 μL downstream primer, 12.5 μL 2×Phanta Max Master Mix, and ddH2O was added to a total reaction volume of 25 μL. The reaction program was: pre-denaturation at 98 °C for 30 s; denaturation at 95 °C for 10 s, annealing at Tm temperature for 5 s, extension at 72 °C for 5 s, 30 cycles of amplification; final extension at 72 °C for 1 min. The reaction products were verified by 1% agarose gel electrophoresis, and the results are shown in Figure 5. WT represents the original MDCK cell line, and KO represents the MDCK cell line with FLNB gene knockout. Theoretically, both homologous chromosomes of MDCK-FLNB-KO cells have inserted a puromycin resistance gene fragment of approximately 1371 bp in length, meaning that the MDCK-FLNB-KO cell band is 1179 bp larger than that of MDCK-WT cells. In the figure, the amplified band of MDCK-WT cells is approximately 2500 bp, and the amplified band of MDCK-FLNB-KO cells is approximately 3700 bp, indicating that homozygous cells with FLNB gene knockout (MDCK-FLNB- / -) were obtained. The PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0086] The primers for cell PCR identification are as follows:

[0087] Upstream primer: 5'-CTCGGATCTCGCCTGGAGTCAA-3'

[0088] Downstream primer: 5'-GAGAGGAACCCGACACCAAC-3'

[0089] Example 2

[0090] This embodiment investigated the transcriptional and protein levels of the FLNB gene knockout MDCK cell line prepared in Example 1.

[0091] 1. qPCR validation of FLNB knockout cells

[0092] qPCR primers were designed based on the FLNB gene and GAPDH internal reference gene, respectively. Total RNA was extracted from MDCK-WT cells and MDCK-FLNB-KO cells using the MiniBEST Universal RNA Extraction Kit (TaKaRa). PrimeScript was used to analyze the RNA. TM cDNA was synthesized by reverse transcription using IV 1st strand cDNA Synthesis Mix (TaKaRa). The reverse transcription reaction system consisted of: total RNA template (100 ng), 4 μL of 5×PrimeScript IV cDNA Synthesis Mix, 1 μL of Random 6mers, and RNase-free dH2O to a total reaction volume of 20 μL. The reaction program was: 30℃ for 10 min; 42℃ for 20 min; 95℃ for 5 min. The reverse transcription product was then subjected to qPCR experiments using an Applied Biosystems 7500 system. The reaction system consisted of: cDNA template (20 ng), 10 μL of 2×ChamQ SYBR qPCR Master Mix (Low ROX Premixed), 1 μL of upstream primer, 1 μL of downstream primer, and ddH2O to a total reaction volume of 20 μL. The reaction program was as follows: pre-denaturation at 95℃ for 30s; cyclic reaction at 95℃ for 10s, 60℃ for 30s, for 40 cycles of amplification; and melting curves were collected.

[0093] The qPCR identification primers are as follows:

[0094] FLNB upstream primer: 5'-TGGAGCGAAGTGGACTGAAGG-3'

[0095] FLNB downstream primer: 5'-GTGTCGTTGGCGTTGTGAATAATG-3'

[0096] GAPDH upstream primer: 5'-CAAGGCTGTGGGCAAGGTCATC-3'

[0097] GAPDH downstream primer: 5'-TTCTCCAGGCGGCAGGTCAG-3'

[0098] The results of qPCR detection of cell mRNA expression level are shown in Figure 6. Compared with MDCK-WT cells, the transcription level of FLNB gene in MDCK-FLNB-KO cell line was reduced by about 95.37% (P < 0.0001).

[0099] 2. Western blot validation of FLNB knockout cells.

[0100] Cells were lysed using RIPA lysis buffer (Shanghai Sangon Biotech), and the supernatant was used for BCA protein quantification (BioSharp). After protein denaturation, proteins were separated using an SDS-PAGE pre-cast gel (GenScript) and then transferred to a nitrocellulose membrane (NC membrane). The membrane was blocked with 5% skim milk at room temperature for 2 hours, washed with PBST, and incubated with primary antibody (FLNB antibody; Abcam; 1:2000) at room temperature for 2 hours. After PBST washing, the membrane was incubated with secondary antibody (HRP-labeled goat anti-rabbit IgG, Shanghai Sangon Biotech; 1:50000) at room temperature for 1 hour. Membrane proteins were stained using ECL reagent (Millipore); scanning and image analysis were performed using an Amersham ImageQuant 800 system (Cytiva, Japan). Western blot results are shown in Figure 7. With GAPDH as an internal control gene, the protein expression level of MDCK-FLNB-KO cells was 97.75% lower than that of MDCK-WT cells (P < 0.001).

[0101] Example 3

[0102] This embodiment studies the growth characteristics and proliferation capacity of the FLNB gene knockout MDCK cell line prepared in Example 1.

[0103] 1. Cell growth curve

[0104] MDCK-WT and MDCK-FLNB-KO cells in logarithmic growth phase were digested with trypsin to form a cell suspension, and then 5 × 10⁶ cells were added to each well. 4 Initially, cells were seeded into six-well plates and cultured at 37°C. Cells were harvested every 24 hours for digestion and counting, a process continued for 10 days. Cell growth curves were plotted with time (h) on the x-axis and cell count on the y-axis. The results are shown in Figure 8. Compared to MDCK-WT cells, MDCK-FLNB-KO cells showed similar cell proliferation capacity, but a slightly larger total cell count at the plateau phase.

[0105] 2. Plate colony formation experiment

[0106] MDCK-WT and MDCK-FLNB-KO cells in logarithmic growth phase were digested with trypsin to form a cell suspension, and then 2 × 10⁶ cells were added to each well. 3 Initially, cells were seeded into six-well plates and cultured at 37°C. Observations were made every 24 hours. After 96 hours of culture, when small cell clusters appeared but did not coalesce, the supernatant was removed, and the cells were washed twice with PBS buffer. The cells were fixed with 4% paraformaldehyde for 20 minutes, followed by staining with 1% crystal violet for 20 minutes. Cell clumping was then observed, and the results are shown in Figure 9. MDCK-WT cells grew densely, forming cell clusters, while MDCK-FLNB-KO cells grew loosely, scattered throughout the culture medium, with a low clumping rate. The number and area of ​​cell clonal clusters were essentially the same for both types of cells.

[0107] 3. Cell passage observation

[0108] When the confluence of MDCK-WT and MDCK-FLNB-KO cells reached 80%, they were passaged at a ratio of 1:4 and cultured at 37°C. Cell characteristics were observed using a cell counter. The results are shown in Figure 10. Compared with MDCK-WT cells, MDCK-FLNB-KO cells had higher viability, slightly smaller diameter, more rounded shape, and lower clumping rate, indicating that the growth morphology of MDCK-FLNB-KO cells has changed.

[0109] Example 4

[0110] This embodiment investigated the adhesion ability of the FLNB gene knockout MDCK cell line prepared in Example 1.

[0111] 1. Cell fluorescence staining

[0112] Cell culture slides were placed in 12-well plates. After removal, the cells were washed three times with PBS buffer, fixed with fixative for 20 min, and washed three times. Then, 200 μL of Actin-Tracker Rad working solution (Beyotime, 1:100) was added to each well, and the cells were incubated at room temperature in the dark for 1 hour. After washing three more times, the cells were placed on slides containing anti-quenched DAPI, sealed, and stored at 4°C for confocal fluorescence imaging. The results are shown in Figure 11. Phallooidin staining was used to stain the cells. Phallooidin specifically binds to actin filaments, clearly showing the morphology and distribution of intracellular actin filaments. Actin filaments interact with integrin receptors in the extracellular matrix, participating in the formation and regulation of focal adhesion. As can be seen from the figure, the actin filament cytoskeleton of MDCK-WT cells is more prominent, while it is almost invisible in MDCK-FLNB-KO cells.

[0113] 2. Cell adhesion experiment

[0114] Following the instructions of the Cell Adhesion Kit (Shanghai Beibo), add the coating solution to a 96-well plate and incubate overnight at 4°C. Before use, remove the coating solution and air dry in a biosafety cabinet for several minutes. Wash three times with washing buffer. Add 10 μL of cells to each well. 4 Cells were cultured at 37°C for 1 hour, the culture medium was removed, and the cells were washed three times with PBS. 100 μL of culture medium and 10 μL of cell staining solution were added, and the cells were incubated at 37°C for 2 hours. OD values ​​were read at 450 nm using a microplate reader. The results are shown in Figure 12. Compared with MDCK-WT cells, the cell-extracellular matrix adhesion ability of MDCK-FLNB-KO cells decreased by approximately 24.3% (P < 0.01).

[0115] 3. Detection of intracellular calcium ion concentration

[0116] According to the instructions for use of the Fluo-4 calcium ion detection kit (Beyotime), use 5 × 10⁻⁶ cells per well. 3 Cells were seeded in 96-well plates and cultured overnight at 37°C. The culture medium was then aspirated, and the cells were washed once with PBS. 100 μL of Fluo-4 staining solution was added to each well, and the cells were incubated at 37°C in the dark for 30 min. Fluorescence analysis was performed using a full-field cell scanning analyzer. The results are shown in Figure 13. Compared with MDCK-WT cells, MDCK-FLNB-KO cells had significantly higher intracellular Ca2+ levels. 2+ The concentration decreased by approximately 41.44% (P < 0.0001), and the decrease in intracellular calcium ion concentration would affect the adhesion strength between cells.

[0117] 4. Cellular trypsin digestion test

[0118] MDCK cells are adherent cells, and trypsin digestion is required during cell passage and expansion. MDCK-WT cells and MDCK-FLNB-KO cells with 95% confluence were digested separately, and the digestion was observed under a microscope. The results are shown in Figure 14. The trypsin digestion time for MDCK-WT cells was 25 minutes, while that for MDCK-FLNB-KO cells was 15 minutes. The reduced trypsin digestion time directly reflects a decrease in cell adhesion ability.

[0119] Example 5

[0120] This embodiment investigated the migration rate and tumorigenicity of the FLNB gene knockout MDCK cell line prepared in Example 1.

[0121] 1. Cell scratch test

[0122] Cells were seeded in streaked six-well plates and cultured overnight until 100% confluence. The culture medium was removed, and the cells were washed with PB. Cells were then streaked with a 10 μL pipette tip, and serum-free medium was added. Cells were cultured at 37°C. Cells were photographed and recorded under a microscope at 0 h, 4 h, 8 h, 12 h, and 24 h. The results were analyzed using ImageJ software. Cell migration rate was calculated as (initial scratch area - scratch area at time t) / initial scratch area. The results are shown in Figure 15. MDCK-WT cells migrated rapidly and almost completely healed the wound at 24 h, with a migration rate of 94.53%. MDCK-FLNB-KO cells migrated more slowly, with a migration rate of 47.69% at 24 h.

[0123] 2. Soft agar colony formation experiment:

[0124] MDCK-WT and MDCK-FLNB-KO cells in logarithmic growth phase were treated into cell suspensions and cultured at 2 × 10⁶ cells per well. 3 In a soft agar colony formation assay, cells were cultured in six-well plates with a lower agar concentration of 0.5% and an upper agar concentration of 0.8%. After 21 days of incubation at 37°C, cell clumping was observed by 1% crystal violet staining, and the average cell clone number was calculated by microscopic photography. The results are shown in Figure 16. In cell suspension culture, the average clone number of MDCK-FLNB-KO cells was lower than that of MDCK-WT cells, indicating a decrease in tumorigenicity of FLNB gene knockout MDCK cells.

[0125] Example 6

[0126] This embodiment investigated the sensitivity of FLNB gene knockout MDCK cells prepared in Example 1 to influenza virus.

[0127] MDCK-WT and MDCK-FLNB-KO cells were cultured to 90% confluence, the culture medium was removed, and the cells were washed with PBS buffer. Virus maintenance medium containing 1 μg / mL recombinant trypsin was added, and four strains of virus, H1N1 (IVR215), H3N2 (IVR237), BV (BVR-26), and BY (BVR-1B), were inoculated at MOI = 0.01. The cells were incubated at 34°C for 72 hours, and the viral supernatant was collected for hemagglutination virus titer detection.

[0128] Add 25 μL of PBS to wells 2-12 of a 96-well microcoagulation plate, and add 50 μL of the virus solution to be tested to well 1. Take 25 μL of the virus solution from well 1 and mix thoroughly in the corresponding well of well 2. Repeat this two-fold dilution process from well 2 to well 12, discarding the 25 μL solution from the last well. Add 25 μL of a suspension containing 1% chicken / guinea pig red blood cells to each well, mix thoroughly, and let stand for 30 min before determining the results. The results are shown in Figure 17. MDCK-FLNB-KO cells showed varying degrees of increased sensitivity to all influenza virus subtypes.

[0129] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0130] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for FLNB gene knockout, characterized in that, Using CRISPR / Cas9 technology, sgRNAs targeting exon 1 of the FLNB gene in MDCK cells were designed to construct FLNB gene knockout vector plasmids 1 and 2. Plasmids 1 and 2 were transfected into MDCK cells to obtain a FLNB gene knockout MDCK cell line. This FLNB gene knockout cell line was named MDCK-FLNB-KO and was deposited at the China Center for Type Culture Collection (CCTCC) on November 20, 2024, with accession number CCTCC NO: C2024391.

2. The method according to claim 1, characterized in that, The nucleotide sequence of the sgRNA is shown in SEQ ID No.

1.

3. The method according to claim 1, characterized in that, Plasmid 1 is pX330-sgRNA plasmid.

4. The method according to claim 1, characterized in that, Plasmid 2 is pY75-5-3 plasmid.

5. The method according to claim 4, characterized in that, The pY75-5-3 plasmid contains a 5' homologous arm and a 3' homologous arm for the sequence recognition site of the sgRNA. The sequence of the 5' homologous arm is shown in SEQ ID No. 2, and the sequence of the 3' homologous arm is shown in SEQ ID No.

3.

6. A FLNB gene knockout MDCK cell line, characterized in that, The FLNB gene knockout MDCK cell line is obtained by the method according to any one of claims 1-5.

7. The FLNB gene knockout MDCK cell line obtained by the method according to any one of claims 1-5, or the FLNB gene knockout MDCK cell line according to claim 6, and its application in the preparation of influenza virus vaccines.

8. The application according to claim 7, characterized in that, The influenza viruses include influenza A virus and / or influenza B virus.

9. The application according to claim 8, characterized in that, The influenza virus includes at least one of the H1N1, H3N2, BV, and BY subtypes of influenza virus.

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