Marc-145 cell strain knocked out of mid1 gene and construction method and application thereof

Abstract

The application discloses a MID1 gene knockout Marc-145 cell strain and a construction method and application thereof, and belongs to the field of molecular biology. The application can reliably obtain a homozygote cell line in which MID1 protein expression is lost by targeting a key site of the MID1 gene through a precisely designed sgRNA. A reliable engineering cell model is provided for subsequent functional research. The application first discloses that MID1 gene knockout can significantly promote the replication of PEDV in Marc-145 cells, and the constructed MID1 gene knockout Marc-145 cell strain can be used as a high-efficiency PEDV proliferation host, directly applied to the large-scale production of PEDV virus antigens, vaccines or carriers, helps to reduce the production cost, and provides a new cell model and strategy basis for antiviral drug screening based on a host target.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to a Marc-145 cell line with MID1 gene knockout, its construction method, and its application. Background Technology

[0002] Porcine epidemic diarrhea virus (PEDV) is a major pathogen causing viral diarrhea in piglets, posing a persistent threat to the global pig industry. Basic research on this virus is crucial for elucidating its pathogenic mechanisms and developing novel prevention and control strategies. Marc-145 cells, a commonly used cell model for virological research, are widely applied to studies on the cellular infection mechanisms, replication processes, and identification of related host factors for viruses such as PEDV. However, the limited replication efficiency of wild-type Marc-145 cells against PEDV leads to prolonged experimental cycles for related molecular and cellular experiments, becoming one of the bottlenecks restricting in-depth basic research on PEDV.

[0003] In recent years, the development of gene-editing technologies such as CRISPR / Cas9 has provided new ideas for enhancing viral replication through genetic modification of host cells. By knocking out genes in the host that may inhibit viral replication, it is hoped to obtain stable and efficient cellular substrates that support viral proliferation. The MID1 gene encodes a protein with E3 ubiquitin ligase activity, and studies have shown that it plays a role in various biological processes, but its impact on coronavirus replication, especially PEDV, remains unclear. Although constructing gene knockout cell lines using CRISPR / Cas9 technology is theoretically feasible, achieving efficient and precise knockout of the MID1 gene in specific species-derived cell lines such as Marc-145 still presents technical challenges, including practical difficulties in sgRNA design, editing efficiency optimization, and clone screening.

[0004] Currently, there is a lack of a systematically validated and efficient technical solution for constructing a MID1 gene knockout Marc-145 cell line that can significantly promote PEDV replication and increase viral yield. Therefore, providing a method for constructing a MID1 gene knockout Marc-145 cell line with clear steps, good reproducibility, and definite effects, and verifying its application potential in enhancing PEDV replication, is of significant technical importance and practical application value for improving vaccine development efficiency, reducing production costs, and promoting related basic research. Summary of the Invention

[0005] The purpose of this invention is to provide a Marc-145 cell line with MID1 gene knockout, its construction method, and its applications, to address the problems existing in the prior art. This invention provides a method for efficiently constructing a Marc-145 cell line with stable MID1 gene knockout. The obtained cell line significantly promotes PEDV replication, providing an efficient cell substrate and a new strategy for low-cost, large-scale production of PEDV vaccines and screening of antiviral drugs.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a method for constructing a Marc-145 cell line with MID1 gene knockout, comprising the following steps: A recombinant vector containing an sgRNA expression cassette targeting the MID1 gene was constructed. The recombinant vector was introduced into Marc-145 cells, and the MID1 gene was knocked out using the recombinant vector. After screening, a Marc-145 cell line with the MID1 gene knocked out was obtained. The target sequence of the MID1 gene is shown in SEQ ID NO.1; The sgRNA is formed by annealing the sense strand with the nucleotide sequence shown in SEQ ID NO.2 and the antisense strand with the nucleotide sequence shown in SEQ ID NO.3.

[0007] Optionally, the recombinant vector is obtained by inserting the sgRNA into the pX459(pSpCas9(BB)-2A-Puro)V2.0 vector at the Bbs I restriction site.

[0008] Optionally, the screening can be performed using puromycin under pressure.

[0009] Optionally, the final concentration of the puromycin is 20 μg / mL.

[0010] The present invention also provides a Marc-145 cell line with MID1 gene knockout obtained by the construction method described above.

[0011] The present invention also provides the application of the MID1 gene knockout Marc-145 cell line in PEDV in vitro proliferation culture.

[0012] The present invention also provides an application of the MID1 gene knockout Marc-145 cell line described above in culturing PEDV with high viral titers.

[0013] The present invention also provides an application of the MID1 gene knockout Marc-145 cell line in the production of PEDV viral antigen, viral vaccine or viral vector. The MID1 gene knockout Marc-145 cell line is used as the production cell for PEDV amplification culture to obtain PEDV virus with higher viral titer.

[0014] The present invention also provides an application of the MID1 gene knockout Marc-145 cell line in screening anti-PEDV drugs, using the MID1 gene knockout Marc-145 cell line as a screening model to evaluate the effect of candidate drugs on PEDV replication.

[0015] The present invention discloses the following technical effects: This invention provides an efficient, specific, and reproducible gene editing method for constructing a stable MID1 gene knockout Marc-145 cell line. By precisely designing sgRNAs targeting key sites in the MID1 gene, combined with an optimized CRISPR / Cas9 vector construction, transfection, puromycin screening, and monoclonal isolation process, homozygous cell lines lacking MID1 protein expression can be reliably obtained. This method has clear steps and overcomes the limitations and instability of traditional screening methods in increasing viral yield, providing a reliable engineered cell model for subsequent functional studies.

[0016] This invention reveals for the first time that MID1 gene knockout can significantly promote PEDV replication in Marc-145 cells. Experiments confirmed that, compared to wild-type cells, MID1-KO cells showed significantly increased viral N protein expression levels, genome copy number, and viral titer after PEDV infection. This indicates that the cell line constructed in this invention can serve as a highly efficient PEDV proliferation host, directly applicable to the large-scale production of PEDV viral antigens, vaccines, or vectors, helping to reduce production costs and providing a new cell model and strategic basis for host-target-based antiviral drug screening. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1To confirm the successful construction of the PX459(pSpCas9(BB)-2A-Puro-sgRNA)V2.0 recombinant vector, which was validated by sequencing, sequencing revealed that an sgRNA sequence was inserted at the BbsI restriction site of the PX459(pSpCas9(BB)-2A-Puro)V2.0 plasmid. Figure 2 The image shows the expression of MID1 protein in the monoclonal cell line screened in Example 1 of this invention and in wild-type Marc-145 cells as detected by Western Blot; where WT: wild-type Marc-145 cells; MID1-KO: Marc-145 cells with MID1 gene knocked out. Figure 3 The images show the gene sequencing results of cell lines that do not express the MID1 gene-encoded protein and wild-type Marc-145 cells in Example 1 of this invention; the rectangular boxes indicate the target locations of the sgRNA sequences, and the red rectangular boxes indicate the base deletion locations. Figure 4 To illustrate the expression of PEDV-N protein in wild-type Marc-145 cells and MID1 gene knockout Marc-145 cells, respectively, Western blot was used to detect the proliferation of PEDV. GAPDH was used as an internal control; WT represented wild-type Marc-145 cells; and MID1-KO represented MID1 gene knockout Marc-145 cells. Figure 5 The graph shows the PEDV genome copy number as measured by quantitative real-time analysis of PEDV proliferation in wild-type Marc-145 cells and MID1 gene knockout Marc-145 cells, respectively. The vertical axis represents the fold change in PEDV genome copy number, and the horizontal axis WT represents wild-type Marc-145 cells; MID1-KO represents MID1 gene knockout Marc-145 cells. This indicates that P < 0.01; Figure 6 To illustrate the expression of PEDV N protein in wild-type Marc-145 cells and MID1 gene knockout Marc-145 cells, respectively, for the proliferation of PEDV, IFA was used to detect the expression of PEDV N protein. Mock represents uninoculated wild-type Marc-145 cells (blank control); WT represents inoculated wild-type Marc-145 cells; and MID1-KO represents inoculated MID1-KO cells. Figure 7 To illustrate the results of PEDV titer determination using wild-type Marc-145 cells and MID1 gene knockout Marc-145 cells for PEDV proliferation, whole virus solutions were collected; the vertical axis represents the PEDV viral titer logarithm. 10 (TCID 50 / mL), the x-axis WT represents wild-type Marc-145 cells; MID1-KO represents Marc-145 cells with the MID1 gene knocked out; This indicates that P < 0.05. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional biochemical methods. Unless otherwise specified, the experimental materials used in the following examples are conventional biochemical reagents. All quantitative results in the following examples were obtained through triplicate experiments.

[0025] Experimental materials: BbsI endonuclease, 10× PNK Buffer, 6× Loading Buffer, T4 PNK, 10× T4 DNA Ligase Buffer, and T4 DNA ligase were purchased from NEB; puromycin was purchased from Thermo Fisher Scientific; the enhanced chemiluminescent substrate ECL Super Kit was purchased from Wuhan Aiboteke Biotechnology Co., Ltd.; the endotoxin-free extraction kit, gel extraction kit, and DNA extraction kit were purchased from OMEGA; horseradish peroxidase-labeled goat anti-rabbit IgG antibody, horseradish peroxidase-labeled goat anti-mouse IgG antibody, and mouse anti-GAPDH monoclonal antibody were purchased from Wuhan Sanying Biotechnology Co., Ltd.; Cy3-labeled goat anti-mouse IgG (H+L) and rabbit anti-MID1 polyclonal antibody were purchased from Wuhan Boster Biological Engineering Co., Ltd.; RIPA protein lysis buffer and DAPI staining agent were purchased from Shanghai Beyotime Biotechnology Co., Ltd.; AceQ qPCR Probe Master Mix, HiScript II Q RT SuperMix for qPCR (+gDNAwiper), and FastPure Cell / Tissue Total RNA Isolation Kit were purchased. V2 and 2 × Phanta Max MasterMix were purchased from Nanjing Novizan Biotechnology Co., Ltd.; pX459(pSpCas9(BB)-2A-Puro)V2.0 vector plasmid was purchased from Beijing Zhongyuan Biotechnology Co., Ltd.; newborn calf serum was purchased from Gibco Biotechnology Co., Ltd.; and high-glucose DMEM complete medium was purchased from Yuanpei Biotechnology Co., Ltd.

[0026] Example 1: Construction of Marc-145 cell line with MID1 gene knockout This embodiment provides a method for constructing a stable knockout cell line of MID1 gene in African green ape kidney epithelial cells (Marc-145 cells) using CRISPR / Cas9 gene editing technology.

[0027] I. Design and Synthesis of sgRNA The MID1 gene sequence (GeneID: 103231585) of the African green ape (Chlorocebus sabaeus) was obtained from the NCBI database, and the intron, exon, and CDS regions of this gene sequence were determined.

[0028] Using the online sgRNA design tool CRISPOR (http: / / crispor.tefor.net / ), we designed, screened, and evaluated off-target effects of sgRNAs targeting the MID1 gene.

[0029] The target site was selected from positions 603 to 622 of the CDS region (with the start codon ATG being +1), and the specific target sequence is 5'-TGGGCGGCACCGCGATCATC-3' (SEQ ID NO.1).

[0030] To facilitate cloning into a CRISPR vector, two complementary oligonucleotide chains were designed and synthesized. The forward strand had a CACC added to the 5' end of the target sequence, and the first T base of the original sequence was replaced with G, resulting in the sgRNA-MID1-F sequence (SEQ ID NO. 2). The reverse strand had AAAC added to the 5' end of the inverse complementary sequence of the target sequence, resulting in the sgRNA-MID1-R sequence.

[0031] sgRNA-MID1-F: 5'- CACC GGGGCGGCACCGCGATCATC-3', SEQ ID NO.2; sgRNA-MID1-R: 5'- AAAC GATGATCGCGGTGCCGCCCC-3', SEQ ID NO. 3.

[0032] II. Construction of CRISPR / Cas9 recombinant plasmids 1. Carrier linearization The pX459(pSpCas9(BB)-2A-Puro)V2.0 plasmid was digested with the restriction endonuclease Bbs I to open its sgRNA cloning site.

[0033] Enzyme digestion system: 3 μg pX459(pSpCas9(BB)-2A-Puro)V2.0 plasmid, 4 μL 10× NEB Buffer, 1 μL Bbs I restriction enzyme, and ddH2O to a total volume of 40 μL.

[0034] Reaction procedure: Enzyme digestion at 37℃ for 3 h.

[0035] Product processing: After the reaction was complete, 6× DNA Loading Buffer was added to terminate the enzyme digestion reaction. The DNA was separated by electrophoresis using a 1% agarose gel, and the target band was excised and recovered using a gel extraction kit.

[0036] 2. Annealing of sgRNA double-stranded oligonucleotides The two synthesized single-stranded oligonucleotides (sgRNA-MID1-F and sgRNA-MID1-R) were annealed to form a double-stranded sgRNA fragment.

[0037] Annealing system: 10× PNK Buffer 1 μL, sgRNA-MID1-F 1 μL, sgRNA-MID1-R 1 μL, T4PNK 0.5 μL, add ddH2O to a total volume of 20 μL.

[0038] Annealing conditions: 37℃ 60 min, 95℃ 5 min, 90℃ 1 min, 85℃ 1 min, 80℃ 1 min, 75℃ 1 min, 70℃ 1 min, 65℃ 1 min, 60℃ 1 min, 55℃ 1 min, 50℃ 1 min, 45℃ 1 min, 40℃ 1 min, 35℃ 1 min, 30℃ 1 min, 25℃ 1 min, 20°C for 1 min, 15°C for 1 min, and store at 10°C.

[0039] 3. Connection reaction The linearized vector was ligated with the annealed sgRNA fragment.

[0040] Ligation system: 1 μL sgRNA annealing product, 30 ng pX459(pSpCas9(BB)-2A-Puro)V2.0 digestion product, 1 μL 10×T4 DNA Ligase Buffer, 0.5 μL T4 DNA Ligase, and ddH2O to bring the volume to 10 μL.

[0041] Connection conditions: Connect at 37℃ for 2 hours.

[0042] After the ligation reaction is complete, the conversion is carried out.

[0043] 4. Transformation 100 μL of Stbl3 chemocompetent cells were removed from -80 °C and allowed to thaw slowly on ice. Then, 10 μL of ligation product was added. The cells were incubated on ice for 30 min, then heat-shocked in a 42 °C water bath for 90 s, and then placed back on ice for 150 s. 900 μL of LB medium was then added, and the cells were cultured at 37 °C and 180 rpm for 1 h. Finally, the cells were transferred to LB culture dishes containing ampicillin resistance (100 μg / mL ampicillin) and spread evenly with a stick. The cells were then incubated at 37 °C for 14 h.

[0044] 5. Screening for target colonies Single colonies were selected and expanded for culture. Identification was performed by PCR and gel electrophoresis. The amplified bands were then sent to Qingke Biotechnology Co., Ltd. for sequencing. Strains with correctly matched sequencing results were expanded for culture, and plasmids were extracted. The recombinant plasmid was named PX459(pSpCas9(BB)-2A-Puro-sgRNA)V2.0. The results are as follows: Figure 1 .

[0045] III. Transfection of Marc-145 cells and selection of MID1 knockout cell lines 1. Cell preparation and transfection Wild-type Marc-145 cells were seeded in 6-well plates and cultured in high-glucose DMEM complete medium containing 10% newborn calf serum at 37 ℃ in a 5% CO2 incubator until the cells reached 80%-90% confluence.

[0046] The recombinant plasmid PX459(pSpCas9(BB)-2A-Puro-sgRNA)V2.0 (experimental group) and the empty vector plasmid PX459(pSpCas9(BB)-2A-Puro)V2.0 (negative control group) were transfected into Marc-145 cells, respectively. A blank control group without any transfection was also set up.

[0047] 2. Puromycin-based pressure screening 24 h after transfection, the culture medium was replaced with complete medium containing puromycin at a final concentration of 20 μg / mL, and the transfected cells were subjected to pressure selection. The blank control group cells were treated with complete medium containing puromycin.

[0048] The culture medium containing puromycin was changed every 3 days, and the selection was continued for 6 days. Cell death was observed and recorded during this period until all cells in the untransfected blank control group died. Recombinant plasmid-transfected cells were cultured until obvious resistant cell clusters formed in the wells.

[0049] 3. Isolation and expansion of monoclonal cells Cells that survived the pressure screening were digested with trypsin, and then diluted to 10 cells / mL using the limiting dilution method. 150 μL of each cell was seeded into a 96-well round-bottom plate and incubated.

[0050] Regularly observe under a microscope, labeling wells containing only a single-cell clone.

[0051] When the monoclonal cells reached a confluence of 50%-70%, they were digested with trypsin and transferred to 24-well plates for further culture. Subsequently, they were transferred to 12-well and 6-well plates for further expansion.

[0052] IV. Identification of MID1 gene knockout 1. Protein level identification (Western Blot) We used Western blotting to detect the expression of MID1 protein in healthy monoclonal cells to be identified and wild-type Marc-145 cells.

[0053] The results are as follows Figure 2 As shown, by Figure 2 It can be seen that the MID1 protein was not expressed in the monoclonal cells to be identified, which suggests that the cells may be monoclonal cell lines with successful MID1 gene knockout.

[0054] 2. Gene-level identification (PCR and sequencing) Cell lines that do not express MID1 protein were selected, and total DNA was extracted using a DNA extraction kit. PCR amplification was performed using identification primers MID1-KO-Check-F and MID1-KO-Check-R.

[0055] MID1-KO-Check-F: 5'-TTGACCTCCCTGTGCCTAATC-3' (SEQ ID NO. 4); MID1-KO-Check-R: 5'-GCCCTCACCTTGACAACACTA-3' (SEQ ID NO. 5).

[0056] PCR system: 12.5 μL of 2 × Phanta Max Master Mix, 1 μL each of primers MID1-KO-Check-F / MID1-KO-Check-R, 2 μL of genomic DNA, and ddH2O to 25 μL.

[0057] PCR program: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30 s, annealing at 60 ℃ for 30 s, extension at 72 ℃ for 1 min, for 30 cycles; final extension at 72 ℃ for 10 min.

[0058] The PCR products were subjected to 1% agarose gel electrophoresis.

[0059] The target band was excised, purified using a gel electrophoresis system, and sent to Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with the wild-type MID1 gene sequence. Sequencing comparison results ( Figure 3 The results showed that the successfully obtained cell line had a 32-nucleotide deletion in the target region (including 15 nt upstream of the target and 1-17 nt at the target), resulting in abnormal MID1 expression, thus successfully knocking out the MID1 gene. The cell line was named MID1-KO-Marc-145.

[0060] Example 2: Effect of MID1 gene knockout on PEDV replication in Marc-145 cells This embodiment aims to evaluate the effect of the constructed MID1-KO-Marc-145 cell line on the replication capacity of porcine epidemic diarrhea virus (PEDV).

[0061] 1. Cell inoculation and viral infection Wild-type Marc-145 cells and MID1-KO-Marc-145 cells were seeded into 12-well plates, 1 × 10⁶ cells per well. 5 Cells were cultured in complete culture medium for 24 hours to achieve a cell density of approximately 90%.

[0062] Discard the culture medium and gently wash the cells three times with PBS.

[0063] The PEDV virus solution was diluted with virus maintenance medium (high glucose DMEM containing 10 μg / mL trypsin), and cells were seeded at MOI=0.1 and incubated at 37 ℃ for 2 h for adsorption.

[0064] After adsorption is complete, discard the virus solution and wash the cells three times with PBS to remove any unadsorbed viruses.

[0065] Add 1 mL of virus maintenance solution to each well and continue culturing in a 37 ℃, 5% CO2 incubator.

[0066] 2. Detection of viral replication level (1) Western Blot detection of viral protein expression Cells were collected 12 hours after virus exposure, and total protein was extracted for Western blotting to detect the expression level of PEDV N protein in the cells. Results are as follows: Figure 4 As shown, the expression level of PEDV N protein was significantly increased in MID1-KO-Marc-145 cells compared with wild-type cells.

[0067] (2) Absolute real-time PCR detection of viral genome copy number RNA extraction and reverse transcription: 12 h after infection, cell culture supernatants were collected, and total RNA was extracted using the FastPure Cell / Tissue Total RNA Isolation Kit V2. After determining the RNA concentration, 1 μg of RNA was used for reverse transcription to synthesize cDNA using the HiScript II QRT SuperMix for qPCR kit. Reverse transcription system: 4 μL of 5×HiScript II qRT SuperMix II, 1 μg of RNA, and RNase-free ddH2O to a final volume of 16 μL; reverse transcription conditions: 55 ℃ for 15 min, 85 ℃ for 5 s.

[0068] qPCR detection: Absolute quantification was performed using AceQ qPCR Probe Master Mix.

[0069] Primers and probes: qPEDV-F: 5'-GGCATTTCTACTACCTCGGA-3', SEQ ID NO.6; qPEDV-R: 5'-CGCCTTCTTTAGCAACCCAG-3', SEQ ID NO.7; qPEDV-probe: 5'-(FAM)-ACCTCACGCCGACCTCCGCT-(BHQ1)-3', SEQ ID NO. 8.

[0070] Reaction system: 10 μL of 2× AceQ qPCR Probe Master Mix, 20.4 μL of 50× ROX Reference Dye, 0.4 μL each of primers qPEDV-F / qPEDV-R, 0.2 μL of probe qPEDV-probe, 4 μL of cDNA template, and ddH2O added to a final volume of 20 μL.

[0071] Reaction program: 95 °C pre-denaturation for 5 min; 95 °C denaturation for 10 s, 60 °C extension for 30 s, for a total of 40 cycles.

[0072] Viral genome copy number was calculated using a standard curve. Results are as follows: Figure 5 As shown, the PEDV genome copy number in the supernatant of MID1-KO-Marc-145 cells was significantly higher than that in the wild-type cell control group.

[0073] (3) Indirect immunofluorescence (IFA) Cells were seeded in 12-well plates and infected with PEDV for 16 h, then fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Staining was performed using anti-PEDV-N protein monoclonal antibody (1:500) as the primary antibody and Cy3-labeled goat anti-mouse IgG (H+L) (1:500) as the secondary antibody, with DAPI counterstaining of the cell nuclei. The proportion of virus-positive cells was observed under a fluorescence microscope. Results are shown below. Figure 6 As shown, the number of PEDV-positive cells in MID1-KO-Marc-145 cells was significantly higher than that in wild-type cells.

[0074] (4) Half-maximal dose of infection (TCID) 50 Detecting viral titers Vero cells were seeded into 96-well plates and cultured until a dense monolayer was formed.

[0075] Cell culture supernatant 12 h after infection was serially diluted 10-fold in eight different levels of maintenance medium (10... -1 Up to 10 -8Each dilution was seeded in 8 replicate wells, with 100 μL per well. Control wells containing only maintenance medium were also included.

[0076] Cells were cultured in a 37°C, 5% CO2 incubator, and the cytopathic effect (CPE) of each well was observed and recorded daily.

[0077] The TCID of the viral fluid was calculated using the Reed-Muench method. 50 The result is as follows Figure 7 As shown, the viral titer produced by MID1-KO-Marc-145 cells (expressed as Log) 10 (TCID 50 The viral load ( / mL) was significantly higher than that of wild-type cells, indicating a significant increase in viral yield. This suggests that the MID1 gene knockout cell line MID1-KO-Marc-145 contributes to PEDV proliferation.

[0078] In summary, this invention successfully constructed a stable MID1 gene knockout Marc-145 cell line (MID1-KO-Marc-145). Virological experiments showed that MID1 gene knockout significantly promoted PEDV replication in Marc-145 cells, manifested as enhanced viral protein expression, increased viral genome copy number, and a substantial increase in viral titer. Therefore, the stable MID1 gene knockout Marc-145 cell line can serve as a highly efficient cell substrate for PEDV proliferation, vaccine production, or related antiviral research.

[0079] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for constructing a Marc-145 cell line with MID1 gene knockout, characterized in that, Includes the following steps: A recombinant vector containing an sgRNA expression cassette targeting the MID1 gene was constructed. The recombinant vector was introduced into Marc-145 cells, and the MID1 gene was knocked out using the recombinant vector. After screening, a Marc-145 cell line with the MID1 gene knocked out was obtained. The target sequence of the MID1 gene is shown in SEQ ID NO.1; The sgRNA is formed by annealing the sense strand with the nucleotide sequence shown in SEQ ID NO.2 and the antisense strand with the nucleotide sequence shown in SEQ ID NO.

3.

2. The construction method according to claim 1, characterized in that, The recombinant vector was obtained by inserting the sgRNA into the pX459(pSpCas9(BB)-2A-Puro)V2.0 vector at the site of BbsI restriction enzyme digestion.

3. The construction method according to claim 1, characterized in that, The screening was performed using puromycin under pressure.

4. The construction method according to claim 3, characterized in that, The final concentration of the puromycin was 20 μg / mL.

5. A Marc-145 cell line with MID1 gene knockout obtained by the construction method according to any one of claims 1-4.

6. The application of the MID1 gene knockout Marc-145 cell line as described in claim 5 in PEDV in vitro proliferation culture.

7. The application of the MID1 gene knockout Marc-145 cell line as described in claim 5 in culturing PEDV with high viral titers.

8. The use of the MID1 gene knockout Marc-145 cell line as described in claim 5 in the production of PEDV viral antigens, viral vaccines, or viral vectors, characterized in that, Using the Marc-145 cell line with MID1 gene knockout as described in claim 5 as the production cell, PEDV amplification culture was performed to obtain PEDV virus with a higher viral titer.

9. The application of the MID1 gene knockout Marc-145 cell line as described in claim 5 in screening anti-PEDV drugs, characterized in that, The MID1 gene knockout Marc-145 cell line was used as a screening model to evaluate the effect of candidate drugs on PEDV replication.

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