SgRNA targeting knockout of p2ry4 gene and application thereof in inhibiting pseudorabies virus replication

Abstract

The application discloses sgRNA for targeting and knocking out a P2RY4 gene and application of the sgRNA in inhibiting replication of pseudorabies virus, and belongs to the field of genetic engineering. The sgRNA is designed according to a P2RY4 gene sequence, and is inserted into a PX459 vector to construct a PX459-sgRNA-P2RY4 recombinant plasmid; the plasmid is electrically transformed into IPEC-J2 cells, and a mutant cell strain with the P2RY4 gene knocked out is obtained through resistance screening; it is found through experiment verification that the mutant cell strain with the P2RY4 gene knocked out has a certain inhibiting effect on infection of pseudorabies virus. Therefore, the application provides a new target for research and development of anti-pseudorabies virus drugs, and has important significance for disease control of the pig raising industry.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, and in particular to sgRNA that targets and knocks out the P2RY4 gene and its application in inhibiting pseudorabies virus replication. Background Technology

[0002] Pseudorabies virus (PRV) has a wide host range, including livestock, wild animals, and laboratory animals such as mice and rabbits. Pigs are the natural host of PRV, and infection is primarily manifested in respiratory symptoms in older pigs, fetal death, and abortion in pregnant sows. In piglets and more susceptible species, PRV infection is often fatal because it causes central nervous system disease. Although PRV infection is rare in humans, it poses a potential threat to public health. Additionally, PRV can cause acute encephalitis, endophthalmitis, and retinal vasculitis in humans. PRV can also establish a lifelong latent infection in the peripheral nervous system. In pigs exposed to external stimuli or with weakened immunity, latent PRV is reactivated from peripheral neurons, replicates, and produces new progeny viruses, leading to the re-emergence of pseudorabies.

[0003] Cells release adenosine triphosphate (ATP) in response to infection or other dangerous stimuli, playing a crucial role in regulating autocrine and paracrine signaling events. ATP, along with other nucleotides, activates two families of purinergic receptors: P2RX and P2RY receptors. P2RY receptors are G protein-coupled receptors (GPCRs) that, depending on their associated heterotrimeric G proteins, induce signaling cascades via different second messengers; they are activated by purines and pyrimidines. Purinergic receptors are expressed in various mammalian cells and are activated by extracellular adenine and uridine nucleotides. P2RY1, P2RY2, and P2RY4 are members of the Y-type purinergic receptor family, promoting astrocyte proliferation and activated by ATP / ADP. P2RY2 regulates important physiological functions, including neurotransmission, inflammation, cell growth, and apoptosis. Recently, it has been reported that the P2 receptor subtype Y1 (P2RY1) in astrocytes inhibits excitatory glutamatergic synaptic neurotransmission. Purinergic type 2 receptors are considered an important component of glial-neuronal transmission, regulating neuronal excitation. The purinergic receptor P2Y4 (P2RY4) is located in the plasma membrane and possesses both G protein-coupled purinergic nucleotide receptor and UTP receptor activities. Studies have shown that it controls many physiological processes and regulates multiple signaling pathways by mediating cellular responses to purines, pyrimidines, and their analogues, such as phospholipase C (PLC) activation, creatine phosphate production, and intracellular calcium regulation. 2+The release of [something] and the synthesis of cAMP, etc. Although P2RY1, P2RY2, and P2RY4 play an inhibitory role in synaptic transmission, postsynaptic P2RY1 and P2RY4 receptors are involved in regulating neurotransmitter release.

[0004] Cells release ATP upon various stimuli. This extracellular ATP release is a prime example of a damage / risk-associated molecular pattern, and numerous studies have demonstrated its crucial role in the immune system. In particular, ATP can activate inflammatory immune responses by binding to and activating purinergic receptors. Studies have reported that IL-1β production in PRV-infected mouse macrophages depends on activation of the NF-κB pathway. Furthermore, during PRV infection, IL-1β secretion depends on ATP-induced purinergic receptor activation and intracellular K+... + The loss of IL-1β and subsequent activation of the NLRP3 inflammasome. PRV infection was found to induce IL-1β expression in macrophages in an NF-κB pathway-dependent manner. Subsequent maturation and secretion of IL-1β are strictly dependent on ATP-purinergic receptor interaction and K+. + Outflow and subsequent NLRP3 activation. Currently, there are no reports of P2RY4 being associated with the PRV infection process. Summary of the Invention

[0005] The purpose of this invention is to provide sgRNA that targets and knocks out the P2RY4 gene and its application in inhibiting pseudorabies virus replication, so as to solve the problems existing in the prior art. By targeting and knocking out the P2RY4 gene, PRV infection in cells can be inhibited, and there is a stronger resistance to PRV. This provides a new target for the development and application of antiviral drugs and is of great significance for disease control in the pig industry.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides the use of sgRNA targeting and knocking out the P2RY4 gene in any of the following:

[0008] (1) Application in the preparation of drugs that inhibit the replication of pseudorabies virus;

[0009] (2) Application in constructing mutant cell lines with P2RY4 gene deletion;

[0010] The nucleotide sequence of the sgRNA is shown in SEQ ID NO.3.

[0011] This invention also provides the application of a gene editing system for targeting and knocking out the P2RY4 gene in any of the following:

[0012] (1) Application in the preparation of drugs that inhibit the replication of pseudorabies virus;

[0013] (2) Application in constructing mutant cell lines with P2RY4 gene deletion;

[0014] The nucleotide sequence of the sgRNA is shown in SEQ ID NO.3.

[0015] Preferably, the nucleotide sequence of the P2RY4 gene is shown in SEQ ID NO.1.

[0016] Preferably, the mutant cell line includes the IPEC-J2 mutant cell line.

[0017] The present invention also provides a method for constructing a P2RY4 gene knockout mutant cell line, comprising the following steps: using the P2RY4 gene as the editing target gene, knocking out the P2RY4 gene in the cell using a gene editing system to construct a P2RY4 gene knockout mutant cell line; wherein, the nucleotide sequence of the sgRNA of the gene editing system is shown in SEQ ID NO.3.

[0018] Preferably, the nucleotide sequence of the edited target gene is as shown in SEQ ID NO.2.

[0019] Preferably, the cells comprise IPEC-J2 cells.

[0020] The present invention also provides a P2RY4 gene knockout mutant cell line, which is prepared using the construction method described above.

[0021] The present invention also provides a drug for inhibiting pseudorabies virus replication, the drug comprising sgRNA for targeting and knocking out the P2RY4 gene, the nucleotide sequence of the sgRNA being shown in SEQ ID NO.3.

[0022] The present invention discloses the following beneficial effects:

[0023] This invention constructed an IPEC-J2 cell line with a P2RY4 gene mutant. CCK-8 assays verified that P2RY4 gene knockout had no effect on the proliferation of this mutant cell line. Quantitative real-time PCR and Western blotting experiments revealed that the P2RY4 gene mutant weakened PRV infection in cells, exhibiting stronger resistance to PRV. The P2RY4 gene can provide a new target for the development of antiviral drugs. As an anti-PRV target, gene knockout or the development of related drugs targeting this target can reduce cell damage and toxicity to mammals such as pigs. Research on PRV is of great significance for disease control in the swine industry. Attached Figure Description

[0024] 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.

[0025] Figure 1 The spectrum of the SpCas9-2A-Puro(PX459)V2.0 vector;

[0026] Figure 2 This is a sequencing map of the PX459 (P2RY4-sgRNA) plasmid.

[0027] Figure 3 This is a sequencing peak diagram of CRISPR target sites in the P2RY4 wild-type IPEC-J2 cell line.

[0028] Figure 4 This is a sequencing peak diagram of CRISPR target sites in the P2RY4 mutant IPEC-J2 cell line (P2RY4-KO1).

[0029] Figure 5 This is a sequencing peak diagram of CRISPR target sites in the P2RY4 mutant IPEC-J2 cell line (P2RY4-KO2).

[0030] Figure 6 Sequence alignment results of P2RY4 wild-type IPEC-J2 cell line and mutant monoclonal cell lines (P2RY4-KO1, P2RY4-KO2); 1: P2RY4 wild-type IPEC-J2 cell; 2: P2RY4-KO1; 3: P2RY4-KO2;

[0031] Figure 7 The results show the cell viability of wild-type P2RY4 IPEC-J2 cells and mutant monoclonal cell lines (P2RY4-KO1, P2RY4-KO-KO2).

[0032] Figure 8 The effects of wild-type P2RY4 IPEC-J2 cell lines and mutant IPEC-J2 cell lines (P2RY4-KO1, P2RY4-KO2) on PRV replication;

[0033] Figure 9 The effects of wild-type P2RY4 IPEC-J2 cell lines and mutant IPEC-J2 cell lines (P2RY4-KO1, P2RY4-KO2) on PRV-gE protein expression were investigated. Detailed Implementation

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] Example 1: Targeted knockout of P2RY4 gene sgRNA and its application in inhibiting pseudorabies virus replication

[0040] 1. Design and synthesize sgRNA

[0041] Based on the obtained P2RY4 gene sequence (NCBI Gene ID: 450244) and CRISPR vector design principles, the target sequence was determined. The target site is located at positions 269–288 of the P2RY4 gene exon region, totaling 20 nucleotides. The GC content should be between 40% and 70%, avoiding four consecutive "T" bases. After removing the 3' PAM sequence NGG, the resulting sequence is: 5'-CGCGTAATAGTAGACAAGGG-3' (SEQ ID NO.2). The sgRNA sequence targeting and editing this DNA sequence is: 5'-CCCUUGUCUACUAUUACGCG-3' (SEQ ID NO.3).

[0042] CACCG was added to the 5' end of the target sequence to form positive Oligo DNA (P2RY4-Oligo-F): 5'-CACCGCGCGTAATAGTAGACAAGGG-3' (SEQ ID NO.4). AAAC was added to the 5' end and C to the 3' end of the reverse complementary DNA sequence of the target sequence to form reverse Oligo DNA (P2RY4-Oligo-R): 5'-AAACCCCTTGTCTACTATTACGCGC-3' (SEQ ID NO.5). The positive and negative strand Oligo DNA (MED13-Oligo-F, R) were then sent to Sangon Biotech for synthesis.

[0043] 2. Construct the PX459(P2RY4-sgRNA) plasmid

[0044] The positive-strand Oligo DNA (P2RY4-Oligo-F) and negative-strand Oligo DNA (P2RY4-Oligo-R) were annealed to form dsDNA. The total reaction volume was 10 μL: 1 μL positive-strand Oligo DNA (P2RY4-Oligo-F) (final concentration 10 μM), 1 μL negative-strand Oligo DNA (P2RY4-Oligo-R) (final concentration 10 μM), 1 μL 10×T4 Ligation Buffer, 0.5 μL T4PNK (T4 polynucleotide kinase, 10 U / μL) (final concentration 5 U), and 6.5 μL ddH2O. The reaction mixture was thoroughly mixed, centrifuged, and then placed in a PCR instrument. The reaction program was as follows: incubation at 37°C for 30 min, annealing at 95°C for 5 min, gradient decreasing to 12°C, 5°C / min.

[0045] The dsDNA formed after annealing was combined with the pSpCas9-2A-Puro(PX459)V2.0 vector (see diagram). Figure 1The ligation system consisted of 10 μL of the following: 25 ng of pSpCas9-2A-Puro(PX459)V2.0 plasmid template, 1 μL of annealed dsDNA, 1 μL of 10×T4 Ligation Buffer, 0.5 μL (2.5 U) of BBSI (5 U / μL), 0.5 μL (200 U) of T4 ligase (400 U / μL), and ddH2O to a total volume of 10 μL. The reaction system was placed in a PCR instrument, and the reaction program was: 37℃ for 5 min, 23℃ for 5 min, for a total of 25 cycles, with the gradient decreasing to 12℃ at 5℃ / min.

[0046] 3. Transformation and screening to identify PX459 (P2RY4-sgRNA) positive clones

[0047] Take a suspension of DH5α competent cells from a -80℃ freezer, dissolve it on ice, aliquot it into 50 μL tubes, add 5 μL of plasmid DNA solution (the ligation product mentioned above), mix well, and place on ice for 30 min; heat shock in a 42℃ water bath for 90 s; cool on ice for 5 min, add 500 μL of ampicillin-free LB liquid medium to the tubes, mix well, and incubate at 37℃ with shaking for 1 h to allow the bacteria to recover to normal growth and express the plasmid-encoded Amp resistance gene; take 100 μL of the above bacterial suspension and spread it evenly on an LB solid culture plate containing Amp, place it upright until the bacterial suspension is completely absorbed by the medium, invert the culture dish and incubate at 37℃ for 16 h.

[0048] Several single colonies were picked using a sterilized pipette tip and inoculated into 1 mL of LB liquid medium containing Amp. The culture was carried out at 37°C and 200 rpm for 2–3 h. PCR was then performed using U6 primers and related antisense oligonucleotides. PCR conditions: 20 μL reaction volume: 1 μL primer (1 ng), 10 μL 2×GS Taq PCR Mix, 9 μL ddH2O. The PCR program was: 94°C pre-denaturation for 3 min; 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1 min, for a total of 34 cycles; final extension at 72°C for 6 min. After PCR identification of positive clones, the samples were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing using the U6 promoter. Sequencing results are as follows: Figure 2 As shown.

[0049] 4. Construction of P2RY4 gene deletion cell lines

[0050] Cells can be arranged at a ratio of 2 × 10 per well. 5 -7×10 5Cells were transferred to new six-well plates and incubated at 37°C with 5% CO2 for 24 h. After 24 h, transfection was performed using Lipo8000™ transfection reagent to transfer the constructed knockout vector into the cell nuclei. Each well of the six-well plate was then replaced with 2 mL of fresh culture medium (DMEM complete medium containing serum and antibiotics) and cultured for another 48 h. After 48 h, once the cells were stable, they were continuously screened using complete medium containing 4 μg / mL puromycin for 7 days, with the medium replaced with fresh puromycin every two days. Subsequently, monoclonal dilutions were performed, and the obtained monoclonal cells were transferred from 96-well plates to 24-well plates for further expansion. Genomic DNA was extracted from different monoclonal cells, primers were designed for regions containing sgRNA, and high-fidelity PCR was performed. The PCR products were sent to Sangon Biotech for sequencing. The PCR primers were P2RY4-F: 5'-CCCTCTGCTTTTGATCGCTG C-3' (SEQ ID NO. 6) and P2RY4-R: 5'-TGACAGCTTCTCAACGAGGTC-3' (SEQ ID NO. 7). The total PCR reaction volume was 20 μL: 0.5 μL P2RY4-F, 0.5 μL P2RY4-R, 10 μL 2×GS Taq PCR Mix, and 9 μL ddH2O. The PCR reaction program was: 94 ℃ pre-denaturation for 3 min; 94 ℃ denaturation for 30 s, 55 ℃ annealing for 30 s, 72 ℃ extension for 1 min, for a total of 34 cycles; final extension at 72 ℃ for 6 min, gradient decreasing to 12 ℃, 5 ℃ / min. The PCR products were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing and analysis. The results are as follows: Figures 3-6 As shown, compared with the wild-type P2RY4 wild-type IPEC-J2 cell line, the target sequence of P2RY4 in the mutant IPEC-J2 cell lines (P2RY4-KO1, P2RY4-KO2) has insertions and deletions, which will produce frameshift mutations and produce non-functional proteins, thus achieving the purpose of gene knockout.

[0051] 5. Effects of wild-type P2RY4 IPEC-J2 cell lines and mutant IPEC-J2 cell lines (P2RY4-KO1, P2RY4-KO2) on PRV replication.

[0052] (1) CCK-8

[0053] The effects of mutant IPEC-J2 cell lines (P2RY4-KO1 and P2RY4-KO2) on cell proliferation activity were detected using the CCK-8 assay. Results are as follows: Figure 7As shown, the difference in absorbance at 450 nm wavelength between the mutant IPEC-J2 cell line and the wild-type IPEC-J2 cell line was not significant (P>0.05), indicating that knocking out the relevant gene had no significant effect on the proliferation of the mutant IPEC-J2 cell line.

[0054] (2) qPCR verification

[0055] After the P2RY4 wild-type IPEC-J2 cell lines and mutant IPEC-J2 cell lines (P2RY4-KO1 and P2RY4-KO2) reached stability, they were inoculated with 0.1 MOI of PRV virus. The supernatant was collected after three freeze-thaw cycles 48 h after infection. Viral DNA was extracted using the Solarbio Animal Cell / Tissue Genomic DNA Extraction Kit and subjected to SYBR Green qPCR. The primers are shown in Table 1 below.

[0056] Table 1 qPCR primers

[0057] like Figure 8 As shown, the results indicate that after 48 h of PRV infection in wild-type IPEC-J2 cell lines and mutant IPEC-J2 cell lines (P2RY4-KO1, P2RY4-KO2), the viral load of PRV in the mutant IPEC-J2 cell lines was significantly lower than that in the wild-type IPEC-J2 cell lines. This is because the P2RY4 gene has been knocked out in the mutant IPEC-J2 cell lines, which affects PRV infection and proliferation on cells, resulting in a lower proliferation level of PRV in the mutant IPEC-J2 cell lines.

[0058] (3) Western Blot Validation

[0059] Wild-type P2RY4 IPEC-J2 cell lines and mutant IPEC-J2 cell lines (P2RY4-KO1 and P2RY4-KO2) were seeded in 6-well plates and infected with PRV. After 48 h, 200 μL of QuickBlock Western primary antibody dilution buffer (purchased from Beyotime) and IP cell lysis buffer were added to each well. After collecting the protein solution, 5× Loading Buffer was added, and the plates were heated at 100℃ for 10 min. The samples were then collected and stored at -20℃. The samples were subjected to polyacrylamide gel electrophoresis, and gel blocks containing the target band were excised and transferred to PVDF membranes. The membranes were blocked with 5% skim milk powder solution for 1.5 h and washed three times with TBST (20 min each time). The plates were then soaked in β-actin mouse monoclonal antibody (5B7) diluted 1:3000 and PRV-gE diluted 1:500, respectively. Monoclonal antibodies were incubated overnight at 4°C and washed three times with TBST (20 min each time). They were then incubated with HRP-goat anti-mouse IgG (H+L) diluted 1:3000 at 60 rpm for 1 h on a shaker and washed three times with TBST (20 min each time). ELC chemiluminescent reagent was added, and the images were then formed using a gel imaging system.

[0060] Imaging results as follows Figure 9 As shown, the results indicate that PRV infection of P2RY4 gene knockout cell lines (P2RY4-KO1, P2RY4-KO2) for 48 h significantly inhibited the expression level of gE protein in PRV. These results demonstrate the successful construction of the P2RY4 gene knockout IPEC-J2 cell line using the method of this invention. The target cell lines P2RY4-KO1 and P2RY4-KO2 constructed in this invention can achieve knockout of the key gene P2RY4, which significantly affects PRV replication intracellularly, thereby significantly improving the cells' resistance to PRV. This lays the foundation for further in-depth research into the molecular mechanism by which the P2RY4 gene inhibits PRV replication and is of great significance for combating pseudorabies virus infection in animal production.

[0061] DNA sequence of the P2RY4 genome in IPEC-J2 cells (SEQ ID NO.1):

[0062]

[0063] 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. Application of sgRNA targeting and knocking out the P2RY4 gene in any of the following: (1) Application in the preparation of drugs that inhibit the replication of pseudorabies virus; (2) Application in constructing mutant cell lines with P2RY4 gene deletion; in, The nucleotide sequence of the sgRNA is shown in SEQ ID NO.3, and the nucleotide sequence of the P2RY4 gene is shown in SEQ ID NO.1; The mutant cell line is the IPEC-J2 mutant cell line.

2. Application of gene editing systems that target and knock out the P2RY4 gene in any of the following: (1) Application in the preparation of drugs that inhibit the replication of pseudorabies virus; (2) Application in constructing mutant cell lines with P2RY4 gene deletion; in, The nucleotide sequence of the sgRNA of the gene editing system is shown in SEQ ID NO.3, and the nucleotide sequence of the P2RY4 gene is shown in SEQ ID NO.1; The mutant cell line is the IPEC-J2 mutant cell line.

3. A method for constructing a P2RY4 gene knockout mutant cell line, characterized in that, Includes the following steps: Using the P2RY4 gene as the target gene for editing, a gene editing system was used to knock out the P2RY4 gene in cells, thereby constructing a P2RY4 gene knockout mutant cell line; wherein, the nucleotide sequence of the sgRNA of the gene editing system is shown in SEQ ID NO.3, and the nucleotide sequence of the P2RY4 gene is shown in SEQ ID NO.1; the cells are IPEC-J2 cells.

4. The construction method as described in claim 3, characterized in that, The nucleotide sequence of the edited target gene is shown in SEQ ID NO.

2.

5. A P2RY4 gene knockout mutant cell line, characterized in that, It is prepared using the construction method described in claim 3 or 4.

6. A drug for inhibiting pseudorabies virus replication, characterized in that, The drug comprises an sgRNA for targeting and knocking out the P2RY4 gene, the nucleotide sequence of which is shown in SEQ ID NO.3, and the nucleotide sequence of which is shown in SEQ ID NO.1.

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