A cell line with tp53 gene knocked out, construction method and use thereof

Abstract

This invention relates to the field of genetic engineering technology, and more particularly to a knockout method. TP53 Cell lines, construction methods, and applications of the gene. This invention provides a cell line with the TP53 gene knocked out, wherein the TP53 gene in this cell line contains a deletion mutation mediated by CRISPR / Cas9. This invention successfully constructs a genetically stable cell line capable of continuous passage. TP53 Gene knockout cell lines. Genome PCR sequencing and Western blot protein detection confirmed the gene knockout at both the nucleic acid and protein levels. TP53 The gene knockout effect was demonstrated, and further, through continuous passaging experiments, it was confirmed that the cell line maintained good genetic stability for at least 20 generations without any reversion mutations, ensuring its reliability in basic research and industrial applications.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more particularly to a knockout method. TP53 Cell lines, construction methods, and applications of genes. Background Technology

[0002] Newcastle disease (ND) is an acute, febrile, and highly contagious infectious disease caused by Newcastle disease virus (NDV), belonging to the family Paramyxoviridae and the genus Avian mumpsvirus. It is widespread globally and is one of the most serious diseases threatening poultry farming. NDV can replicate in HeLa, DF-1, Vero, LMH, CEF, and BHK-21 cells, causing syncytial and other lesions, and inducing apoptosis in infected cells. A single virus can replicate in just 10 hours.

[0003] transcription factors TP53 Its related genes P63 and P73 are important anti-tumor genes, among which TP53 It can play an important role in a variety of cellular responses. Firstly, TP53 Proteins can regulate the cell division cycle and are highly expressed. TP53 Proteins can significantly inhibit cell division; secondly, when cell genes are severely damaged, TP53 Proteins can induce apoptosis; furthermore, TP53 Proteins can also inhibit angiogenesis and have an inhibitory effect on the development of malignant tumors; in addition, TP53 Proteins play an important role in the body's immune regulation, metabolic regulation, and antiviral activity.

[0004] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-Cas) and their associated proteins are an adaptive antiviral immune system found in prokaryotes and most bacteria. They are transcribed into small RNA guides via specific spacer sequences, directing Cas nucleases to specifically cleave viral DNA / RNA. Currently, this technology is widely used in genome editing, gene transcription regulation, and epigenetic engineering, representing one of the greatest scientific achievements of the past decade. Based on the number of Cas effector proteins, CRISPR-Cas systems can be divided into two main categories, further subdivided into six main types (Types I-VI) and 19 subtypes. Type I CRISPR systems utilize multiple Cas protein subunits and crRNA (CRISPR-derived RNA) to form an effector complex, resulting in a more complex cleavage mechanism. The Cas protein composition mainly includes types I, III, and IV. Type 2 CRISPR systems utilize a large, single-component Cas effector protein that binds to crRNA to exert interference and other effects. Only a single Cas protein is needed for target recognition and cleavage, and these systems mainly include type II, V, and VI Cas proteins. Among these, the three most widely used proteins are type II Cas9, type V Cas12, and type VI.

[0005] The efficient replication and cytopathic effect of Newcastle disease virus (NDV) in chickens are closely related to the regulation of host cell apoptosis pathways, providing a starting point for studying the role of host factors in infection. TP53 As a key cellular regulatory hub, its antiviral and immunomodulatory functions suggest it may be a crucial node in virus-host interactions. CRISPR-Cas technology, represented by Cas9 and Cas12, serves as a highly efficient gene-editing tool, enabling targeted gene editing at the cellular level. TP53 This provides powerful tools for understanding functional genes. The convergence of this background knowledge lays the theoretical foundation for using gene editing technology to modify host cells, thereby exploring viral replication mechanisms and developing efficient vaccine production platforms. Summary of the Invention

[0006] The purpose of this invention is to provide a knockout TP53 Cell lines, construction methods, and applications of genes.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a knockout TP53 The cell line of the gene, the cell line TP53The gene contains a deletion mutation mediated by CRISPR / Cas9.

[0008] Preferably, the deletion mutation TP53 The amino acid sequence of the gene is shown in SEQ ID NO:4.

[0009] Preferably, the cell line is BHK-21.

[0010] This invention provides a method for knocking out cells TP53 The sgRNA of the gene, the target sequence of which is shown in SEQ ID NO:1.

[0011] This invention provides a method for constructing the aforementioned cell line, comprising the following steps: (1) Design and synthesize targets TP53 The sgRNA of the gene, whose target sequence is shown in SEQ ID NO:1; (2) The sgRNA and Cas9 protein were co-transfected into BHK-21 cells; (3) Obtain transfected positive cells by drug screening, and obtain single-cell clones by monoclonal screening; (4) Perform treatment on monoclonal cells TP53 Gene knockout identification.

[0012] Preferably, the identification in step (4) includes: (a) PCR amplification using primer pairs, and detection TP53 Gene fragments; (b) Sequencing the PCR products to confirm the type of gene editing; (c) Detection by Western blot TP53 Protein expression level.

[0013] Preferably, the nucleotide sequences of the primer pair are shown in SEQ ID NO:7 and 8.

[0014] This invention provides the application of the aforementioned cell line in the propagation of viruses.

[0015] Preferably, the virus is a single-stranded RNA virus; the single-stranded RNA virus is Newcastle disease virus.

[0016] This invention provides a method for increasing the titer of Newcastle disease virus in chickens in cell culture, using the cell line described above for virus inoculation and culture.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for knocking out hamster kidney cells (BHK-21) using CRISPR / Cas9 technology. TP53 A complete approach to genes was developed, and a genetically stable and continuously passable gene pool was successfully constructed. TP53 Gene knockout cell line BHK-TP53 — This method includes not only treatments for golden hamsters. TP53 The study included the design and validation of gene sequence-specific sgRNAs, efficient transfection strategies, and the establishment of a standardized workflow from drug screening and monoclonal isolation to multi-level identification. Genomic PCR sequencing and Western blot protein detection provided dual confirmation at both the nucleic acid and protein levels. TP53 The gene knockout effect was demonstrated, and further, through continuous passage experiments, it was confirmed that the cell line maintained good genetic stability for at least 20 generations without any reversion mutations, ensuring its reliability in basic research and industrial applications.

[0018] In terms of biological characteristics, BHK-TP53 — The cells showed no significant differences from the parental BHK-21 cells in basic traits such as morphology and growth rate, indicating that... TP53 The gene deletion did not negatively affect the normal adherent growth and proliferation ability of BHK-TP53 cells. After extending the culture time, — The cells exhibited higher cell viability. This result functionally confirms that knockout... TP53 The gene effectively inhibited the apoptosis pathway mediated by it, thereby delaying the aging and death process of cells in the later stages of passage.

[0019] The core application value of this invention lies in significantly improving the proliferation efficiency of Newcastle disease virus (NDV) in cell culture systems. Experimental data show that using BHK-TP53... — Cell culture of NDV Lasota strain, the HA titer and TCID of the harvested virus 50 The titers were significantly higher than those of conventional BHK-21 cells. This demonstrates that regulating the host cell's antiviral apoptosis response through gene editing can effectively remove some of the restrictions on viral replication and create a more favorable intracellular environment for viral amplification. Therefore, BHK-TP53... — As a novel production platform with optimized performance, cell lines are not only suitable for the efficient preparation of NDV vaccine strains, but also provide new technical ideas and cellular tools for the development of production processes for other similar viruses. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0021] Figure 1 BHK-TP53 — Cell clone PCR identification results.

[0022] Figure 2 BHK-TP53 — Cell cloning TP53 Gene sequencing analysis.

[0023] Figure 3 BHK-TP53 — Cellular Western blot identification.

[0024] Figure 4 BHK-TP53 — Cell morphology observation (200×).

[0025] Figure 5 BHK-TP53 — Determination of cell proliferation capacity.

[0026] Figure 6 BHK-TP53 — Assay for cell viability. Detailed Implementation

[0027] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0028] Example 1

[0029] 1. Materials

[0030] 1.1 Biological materials

[0031] NDV Lasota strain (virus content 1×10⁻⁶) 9.0 EID 50 / 0.1 mL HA ratio is 1:2 10 PX459 vector and BHK-21 cells were preserved by the College of Life Sciences, Cangzhou Normal University.

[0032] 1.2 Main Reagents

[0033] DMEM culture medium was purchased from Gibco; fetal bovine serum was purchased from Sijiqing Biotechnology Co., Ltd.; the genome extraction kit was a product of AXYGEN; DNA Marker was purchased from Takara Bio Engineering (Dalian) Co., Ltd.; puromycin was purchased from Beijing Solarbio Technology Co., Ltd.; Cas9 protein was purchased from Genescript; and the CRISPR RNP transfection kit was purchased from Origene.

[0034] 2. Experimental Methods

[0035] 2.1 BHK-21 cells TP53 sgRNA and identification primer design

[0036] According to the golden hamster registered in NCBI TP53 The gene sequence (GeneID 101833915) was designed using CRISPR design software (http: / / crispr.mit.edu / ; http: / / www.e-crisp.org / index.html). TP53 The sgRNA of the gene was identified and named sgRNA-P53 (see Table 1), with the restriction enzyme site BbsI. Simultaneously, a gene was designed and synthesized. TP53 Gene transcription primers and identification primers.

[0037] Table 1 TP53 Gene sgRNA and transcription primers

[0038] As shown in SEQ ID NO:5, 6, 7 and 8

[0039] 2.2 Preparation of PX459-TP53-sgRNA plasmid

[0040] sgRNA-P53 was transcribed using transcription primers and a kit, then ligated into the vector PX459. Following transformation, plasmid extraction and identification, PX459- was prepared. TP53 -sgRNA plasmid.

[0041] 2.3 Transfection of PX459-TP53-sgRNA plasmid

[0042] BHK-21 cells were passaged into 24-well plates using standard methods, and transfection was performed when the cell density reached 90%. First, 5.0 μL of Cas9 protein was placed in a 1.5 mL centrifuge tube, followed by 5.0 μL of PX459-TP53-sgRNA plasmid. The mixture was incubated at room temperature for 10 min, and then 40 μL of CRISPR buffer was added. In another centrifuge tube, 0.8 μL of Viromer and 49.2 μL of CRISPR buffer were added respectively. The liquids in both centrifuge tubes were mixed thoroughly, incubated at room temperature for 15 min, and then transferred to 24-well plates. The plates were then incubated at 37 ℃ in a 5% CO2 incubator.

[0043] 2.4 Drug screening for transfecting BHK-21 cells

[0044] 48 h after transfection with BHK-21 cells, the culture medium was discarded and replaced with a medium containing puromycin (2 μg / mL). Cell growth was observed daily, and the medium was replaced if a large number of cells died and floated. After 14 days of culture, puromycin-free complete medium was added, and the cells were cultured until they filled 24-well plates.

[0045] 2.5 BHK-TP53 — Screening of cell monoclonal antibodies

[0046] Cells were counted in each well, and the cells were infinitely diluted according to the cell count. The resulting solution was added to 96 wells, and complete culture medium was added. The wells were then incubated at 37 ℃ in a 5% CO2 incubator. Wells with single cells were selected and cultured to a monolayer and then expanded.

[0047] 2.6 BHK-TP53 — Identification of cell monoclonal antibodies

[0048] Extract the above BHK-TP53 respectively — The genome of a single cell clone was analyzed using sequencing primers. TP53 PCR amplification of the gene, the target fragment is 696 bp ( Figure 1 The PCR products were then sent to General Biotechnology (Anhui) Co., Ltd. for sequencing. The results showed that one knockout strain was obtained. TP53 BHK-TP53 gene — Single cell clone. Compared with BHK-21 wild-type cells. TP53 Compared to genes, BHK-TP53 — cell TP53 A gene mutation occurred at the 284bp gRNA position, changing from the original TCCGAGAA to CAAC; additionally, at the 340bp position, the original A gene mutated to G (…). Figure 2Finally, the sequencing-positive BHK-TP53 was... — Cellular processes TP53 Western blot analysis of the protein. Results showed that BHK-TP53... — cells TP53 The expression level of the protein was significantly lower in BHK-21 cells than in BHK-21 cells, with the expression level corresponding to the target fragment being lower. TP53 The amino acid sequence of the protein is shown in SEQ ID NO:4. Figure 3 ).

[0049] The PCR reaction system consisted of 20 μL, including 10 μL of 2×buffer, 1.5 μL each of forward and reverse primers, 1.0 μL of Taq enzyme, 4.0 μL of template DNA, and 2.0 μL of water.

[0050] The PCR reaction conditions were: 95 ℃ for 2 min, 95 ℃ for 30 s, 55 ℃ for 30 s, 72 ℃ for 30 s, 36 cycles, followed by a 10 min extension at 72 ℃.

[0051] The nucleotide sequence shown in SEQ ID NO:1 is the original gene sequence.

[0052] The following is a 696bp nucleotide sequence: TGAATTTGGGAAGCGGAGGCAGGGGGATCCAAAGTTTGGAATCACCCTTGACTGGCATCAGTGAGATCCTGTGTACAAAAAAGGAAACCAACAGTGCTAACTCTGTTCTTGCCTTGTTTTGCAGACTTCCTCCAAACAATGTTCTGGTAAGGACCAGGGCGGGAAGGGACTGAGGGTCTGGTGGGTGGCTCTGCTGGGAGACCAAAAGCACAGCACTGAGTCTCTTTTGGCCCCTTCACAGTCCACCTTGCCGTCCTCTGATTCCATTGAAGAACTGTTCCTGTCCGAGAATGTTGCAGGCTGGCTAGAAGACCCAGGTGAAGCTCTCCAAGGGTCGGCAGCTGCGGCAGCGCCGGCGGCTCCTGCAGCAGAGGACCCTGTAGCTGAGACTCCTGCACCGGTGGCCTCTGCGCCAGCCACTCCCTGGCCCCTCTCATCTTCTGTCCCATCCTATAAAACCTACCAGGGCGACTATGGTTTCCGTCTGGGCTTCCTGCACTCGGGGACGGCCAAATCTGTCACATGCACGGTCAGTGGCCCCGAGGTGTTGACTTTCATGTGACTTTTCAACGTCTGTTTTTTTCTGTCAAGATTTTGGGGTTCCTCTTTAGCCCGTGAACTTTGGTGTATGTCTCAGAGTTTAATTTCCTCCTGGACCTTTGCCTTTTATCCTTCTCTCCTGTCCTGTGCATCTCT The following is the corresponding original amino acid sequence (as shown in SEQ ID NO: 2): EFGKRRQGDPKFGITLDWHQ-DPVYKKGNQQC-LCSCLVLQTSSKQCSGKDQGGKGLRVWWVALLGDQKHSTESLLAPSQSTLPSSDSIEELFLSENVAGWLEDPGEALQGSAAAAAPAAPAAEDPVAETPAPVASAPATPWPLSSSVPSYKTYQGDYGFRLGFLHSGTAKSVTCTVSGPEVLTFM-LFNVCFFLSRFWGSSLARELWCMSQSLISSWTFAFYPSLLSCASL The following is the 692bp nucleotide sequence after mutation (the bolded part is the mutated nucleotide sequence; as shown in SEQ ID NO: 3): TGAATTTGGGAAGCGGAGGCAGGGGGATCCAAAGTTTGGAATCACCCTTGACTGGCATCAGTGAGATCCTGTGTACAAAAAAGGAAACCAACAGTGCTAACTCTGTTCTTGCCTTGTTTTGCAGACTTCCTCCAAACAATGTTCTGGTAAGGACCAGGGCGGGAAGGGACTGA GGGTCTGGTGGGTGGCTCTGCTGGGAGACCAAAAGCACAGCACTGAGTCTCTTTTGGCCCCTTCACAGTCCACCTTGCCGTCCTCTGATTCCATTGAAGAACTGTTCCTGCAACTGTTGCAGGCTGGCTAGAAGACCCAGGTGAAGCTCTCCAAGGGTCGGCGGCTGCGGCAG CGCCGGCGGCTCCTGCAGCAGGAGGACCTGTAGCTGAGACTCCTGCACCGGTGGCCTCTGCGCCAGCCACTCCCTGGCCCCTCTCATCTTCTGTCCCATCCTATAAAACCTACCAGGGCGACTATGGTTTCCGTCTGGGCTTCCTGCACTCGGGGACGGCCAAATCTGTCACA TGCACGGTCAGTGGCCCCGAGGTGTTGACTTTCATGTGACTTTTCAACGTCTGTTTTTTTCTGTCAAGATTTTGGGGTTCCTCTTTAGCCCGTGAACTTTGGTGTATGTCTCAGAGTTTAATTTCCTCCTGGACCTTTTGCCTTTTATCCTTCTCTCCTGTCCTGTGCATCTCT The following is the mutated amino acid sequence (the bolded part is the mutated sequence; as shown in SEQ ID NO: 4): EFGKRRQGDPKFGITLDWHQ-DPVYKKGNQQC-LCSCLVLQTSSKQCSGKDQGGKGLRVWWVALLGDQKHSTESSLLAPSQSTLPSSDSIEELFLQLLQAG-KTQVKLSKGRRLRQ RRRLLQQRTL-LRLLHRWPLRQPLPGPSHLLSHPIKPTRATMVSVWASCTRGRPNLSHARSVAPRC-LSCDFSTSVFFCQDFGVPL-PVNFGVCLRV-FPPGPLPFILLSCPVHLS 2.7 BHK-TP53 — Determination of cell growth characteristics (1) BHK-TP53 — Cell morphology observation 8.0×10 6 BHK-TP53 — Cell clones and BHK-21 cells were seeded separately into T25 culture flasks, and DEME medium containing 6% fetal bovine serum was added. After 48 h of culture, the cells were passaged and continuously cultured up to the 5th generation. During this period, BHK-TP53 cells were observed and compared. — Cell clones and the morphology and growth rate of BHK-21 cells. Results showed that BHK-TP53... — The growth rate and morphology of the cells were not significantly different from those of BHK-21 cells. Figure 4 ).

[0053] (2) BHK-TP53 — Cell growth kinetics analysis

[0054] In BHK-TP53 — During continuous passage culture of BHK-21 cells, samples were taken at 24 h, 48 h, 72 h, and 96 h. 10 μL of cell suspension was stained with trypan blue to determine cell number and viability, thus evaluating BHK-TP53. — Cell growth characteristics. Results showed that after 24–48 h of culture, BHK-TP53… — The number of both BHK-TP53 and BHK-21 cells gradually increased, with BHK-TP53 showing the highest growth rate. — The cell count can reach 2.4 × 10⁻⁶. 7 , with BHK-21 cells (density up to 2.36 × 10⁻⁶) 7 The differences were not significant; furthermore, the viability of both cell types remained above 98%, with no significant difference. After culturing for 72-96 hours, BHK-21 cells and BHK-TP53 cells showed... —The number of cells increased in all cases, but the increase was less than 24–48 h; among them, BHK-TP53 — The cell count reached 2.68 × 10⁻⁶. 7 The density of BHK-21 cells can reach 2.55 × 10⁶. 7 In terms of cell viability, BHK-TP53 — Cell viability was 95% at 72 h and remained above 90% at 96 h; while the cell viability of BHK-21 cells was significantly lower than that of BHK-TP53 cells from 72 to 96 h. — Cells. This result may be related to knockout. TP53 This gene is associated with reducing apoptosis. Therefore, BHK-TP53 — The cell passage time is 48–72 h. Figure 5 , Figure 6 ).

[0055] 2.8 BHK-TP53 — Cellular genetic stability test

[0056] BHK-TP53 — Cells were passaged as usual, and samples were taken at F5, F10, and F20 generations to extract the genome for further analysis. TP53 PCR amplification and sequencing analysis of the gene were used to identify BHK-TP53. — The genetic stability of cells. The results indicate that BHK-TP53... — No reversion mutations occurred in the F5, F10, and F20 generations of cells, indicating good genetic stability.

[0057] 2.9 BHK-TP53 — Application of NDV Lasota strain in cell proliferation culture

[0058] (1) NDV Lasota strain inoculated with BHK-TP53 — cell

[0059] BHK-TP 5 3 — Cells and BHK-21 cells were passaged into 6-well cell culture plates using standard methods, 1×10⁶ cells / well. 6Cells were cultured at 1000 cells / well until they grew into a monolayer. The cell surface was washed twice with serum-free DMEM medium, and 10-fold diluted NDV Lasota strain was added at a viral infection dose of 0.5 MOI. The cells were incubated at 37 ℃ for 1 h in a 5% CO2 incubator. The viral fluid was then aspirated, and 2.0 ml / well of serum-free DMEM medium containing 3 μg / mL TPCK-trypsin was added. The cells were incubated at 33 ℃ in a 5% CO2 incubator. Virus was harvested when 80-90% cytotoxicity was achieved and stored at -20 ℃. Results showed that BHK-TP53... — After inoculation, the cell harvesting time was 3–4 hours longer than that of BHK-21 cells.

[0060] (2) HA titer and TCID in cell cultures 50 Measurement

[0061] The above NDV cell cultures were repeatedly frozen and thawed three times to determine HA titer and TCID. 50 The results of the measurement showed that BHK-TP53 — The virus titer in the cell culture medium was 1:2, and the HA titer was 1:2. 10 7.0 lgTCID 50 / 0.1mL, significantly higher than BHK-21 cell culture (Table 2).

[0062] Table 2. BHK-TP53 — HA titer and TCID after cell inoculation with NDV Lasota strain 50 Measurement

[0063] 3. The BHK-TP53 constructed in this invention — The cells are BHK-21 cells based on CRISPR / Cas technology. TP53 It is obtained by knocking out genes and has good stability.

[0064] 4. In summary, the BHK-TP53 constructed using this invention... — Cell proliferation culture of the NDV Lasota strain showed a significantly increased viral titer of 7.0 lgTCID compared to conventional BHK-21 cells. 50 / 0.1mL.

[0065] The BHK-TP53 constructed in this invention — While cell lines can be used to proliferate and culture NDV Lasota strains, they are not limited to the culture of NDV Lasota strains and the preparation of related vaccines.

[0066] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A cell line with the TP53 gene knocked out, characterized in that, The TP53 gene in this cell line contains a deletion mutation mediated by CRISPR / Cas9.

2. The cell line according to claim 1, characterized in that, The amino acid sequence of the deleted TP53 gene is shown in SEQ ID NO:

4.

3. The cell line according to claim 1, characterized in that, The cell line was BHK-21.

4. An sgRNA for knocking out the TP53 gene in cells, characterized in that, The target sequence of the sgRNA is shown in SEQ ID NO:

1.

5. A method for constructing the cell line according to any one of claims 1 to 3, characterized in that, Includes the following steps: (1) Design and synthesize sgRNA targeting the TP53 gene, the targeting sequence of which is shown in SEQ ID NO:1; (2) The sgRNA and Cas9 protein were co-transfected into BHK-21 cells; (3) Obtain transfected positive cells by drug screening, and obtain single-cell clones by monoclonal screening; (4) Identification of TP53 gene knockout in monoclonal cells.

6. The method according to claim 5, characterized in that, The identification in step (4) includes: (a) PCR amplification using primer pairs to detect the TP53 gene fragment; (b) Sequencing the PCR products to confirm the type of gene editing; (c) TP53 protein expression level was detected by Western blot.

7. The method according to claim 6, characterized in that, The nucleotide sequences of the primer pairs are shown in SEQ ID NO:7 and 8.

8. The use of the cell line according to any one of claims 1 to 3 in the propagation of viruses.

9. The application according to claim 8, characterized in that, The virus is a single-stranded RNA virus; the single-stranded RNA virus is Newcastle disease virus.

10. A method for increasing the titer of Newcastle disease virus in chickens during cell culture, characterized in that, Virus inoculation and culture were performed using the cell lines described in any one of claims 1 to 3.

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