Application of ncam2 as a target in screening drugs for inhibiting sindbis virus replication
Patent Information
- Application Number
- CN202510420324.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-03
- Publication Date
- 2026-10-16
AI Technical Summary
[0033]本发明的有益效果是：①本发明首先发现在HEK-293T细胞中敲除NCAM2后能够抑制SVA的复制，可作为靶点用于制备抑制SVA复制的药物；②本发明提供了一种靶向NCAM2基因的sgRNA，所述sgRNA能够特异性靶向NCAM2基因，结合CRISPR-Cas9技术可实现宿主细胞中NCAM2基因的敲除，打靶准确、敲除效率高；③本发明提供了一种通过CRISPR-Cas9技术将所述sgRNA转染于宿主细胞，构建NCAM2基因敲除细胞系的方法，通过使NCAM2基因敲除，获得的了具有SVA抗性表型的细胞系，SVA在所述细胞系中的复制显著受到抑制，病毒核酸和滴度明显降低；能够显著抑制SVA的复制，为进一步研究NCAM2基因在细胞内调控病原微生物复制的分子机制提供研究工具和材料，也可用于抗SVA的动物育种，对未来预防或抑制SVA感染提供一些理论依据。
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of genetic engineering, specifically relating to the application of NCAM2 as a target in screening drugs that inhibit Seneca virus replication. Background Technology
[0002] Senecavirus A (SVA) is the only member of the genus Senecavirus in the family Picornaviridae. It is a newly emerging infectious disease pathogen in pigs. The vesicular disease caused by SVA infection is very similar to the lesions caused by foot-and-mouth disease (FMD), vesicular stomatitis (SVD), and vesicular stomatitis (VS). The clinical symptoms of all three diseases are obvious vesicles and erosions on the snout and hooves of the affected animals, accompanied by lameness, anorexia, lethargy, and fever, which seriously hinders the prevention and control of vesicular disease.
[0003] Developing effective diagnostic and control strategies to prevent the spread of this disease is an urgent problem to be solved. Therefore, a deeper understanding of the mechanisms by which host proteins inhibit SVA replication and the construction of cell lines that inhibit SVA viral replication using CRISPR-Cas9 technology are of great significance for the creation of Seneca virus vaccines.
[0004] NCAM2, also known as olfactory cell adhesion molecule or Rb-8 neural cell adhesion molecule, is a type of cell adhesion molecule (CAM). NCAM2 belongs to the NCAM family of immunoglobulins. In mammals, this family includes two subtypes, NCAM1 and NCAM2, which share the same extracellular domains, including five N-terminal immunoglobulin domains (Ig) and two fibronectin type III domains (Fn3). Many members of the CAM family have been identified as viral receptors; for example, NCAM1 is the receptor for rabies virus and Zika virus. Previous studies have found that NCAM2 expression is upregulated in prostate and breast cancer cell lines; therefore, NCAM2 is considered a potential target for prostate and breast cancer therapy.
[0005] An unexpected study in this invention revealed that knocking out the NCAM2 gene in HEK-293T cells significantly inhibited SVA replication in HEK-293T cells, and the viral nucleic acid and titer were significantly reduced. This can serve as a target for screening drugs that inhibit SVA virus infection, providing important guidance for the prevention and control of SVA virus. Summary of the Invention
[0006] In response to the aforementioned technical problems, this invention unexpectedly discovered that knocking out the NCAM2 gene in HEK-293T cells significantly inhibited SVA replication in HEK-293T cells, and the viral nucleic acid and titer were significantly reduced. This can be used as a target for screening drugs that inhibit SVA virus infection, providing some theoretical basis for the infection mechanism and prevention and control strategies of SVA.
[0007] Specifically, it includes the following:
[0008] In a first aspect, the present invention provides an application of NCAM2 gene / protein as a target in screening drugs for the prevention or treatment of microRNAviridae virus infections; the drug targets NCAM2 gene / protein to inhibit or silence the expression of NCAM2 gene / protein.
[0009] Preferably, the microRNAviridae virus is Seneca virus.
[0010] Secondly, the present invention provides the use of an NCAM2 gene / protein expression inhibitor in the preparation of drugs, pharmaceutical compositions or vaccine compositions for the prevention or treatment of small RNA virus family viral infections.
[0011] Preferably, the NCAM2 gene / protein expression inhibitor includes a small molecule compound that inhibits NCAM2 gene expression, or a small interfering RNA designed to target the NCAM2 gene, or an sgRNA that targets and knocks out the NCAM2 gene.
[0012] Preferably, the sgRNA targets at least one of the following target sequences:
[0013] NCAM2-sgRNA1: acagggtagtagtgcaaa;
[0014] NCAM2-sgRNA2:ggtgaacctgaaagtatagat.
[0015] Preferably, the microRNAviridae virus is Seneca virus.
[0016] Thirdly, the present invention provides an sgRNA that specifically targets and knocks out the NCAM2 gene, wherein the sgRNA targets at least one of the following target sequences:
[0017] NCAM2-sgRNA1: acagggtagtagtgcaaa;
[0018] NCAM2-sgRNA2:ggtgaacctgaaagtatagat.
[0019] Fourthly, the present invention provides the application of the sgRNA described in the third aspect above in the preparation of drugs for the prevention or treatment of microRNA virus infections, or in NCAM2 gene knockout cell lines.
[0020] Preferably, the microRNAviridae virus is Seneca virus.
[0021] Fifthly, this invention provides an application of NCAM2 gene knockout cell lines in breeding for resistance to Picornaviridae viruses.
[0022] Preferably, the microRNAviridae virus is Seneca virus.
[0023] Preferably, the cell line is HEK-293T cells.
[0024] Fifthly, the present invention provides a method for constructing NCAM2 gene knockout HEK-293T cells, the method comprising the following steps:
[0025] (1) Prepare the sgRNA that specifically targets the NCAM2 gene as described in the third aspect above, add a CACC sticky end to the 5' end of the forward sequence of the sgRNA fragment and add an AAAC sticky end to the 5' end of the reverse sequence to serve as an sgRNA oligonucleotide targeting the NCAM2 gene.
[0026] (2) Insert the double-stranded fragment prepared in step (1) into the multiple cloning site of the PX459 expression plasmid vector to obtain a recombinant vector that simultaneously expresses the Cas9 protein gene and the targeting sgRNA sequence.
[0027] (3) Transfect HEK-293T cells with the recombinant vector prepared in step (2), screen and kill negative cells with puromycin antibiotic, pick single cells, seed and culture to obtain NCAM2 gene loss-of-function cell line.
[0028] Preferably, the upstream and downstream primers of the sgRNA are:
[0029] NCAM2-sgRNA1-F:caccgacagggtagtagtgcaaa,
[0030] NCAM2-sgRNA1-R:aaacttgcactactaccctctgtc;
[0031] NCAM2-sgRNA2-F:caccggtgaacctgaaagtatagat,
[0032] NCAM2-sgRNA2-R:aaacatctatactttcaggttcacc.
[0033] The beneficial effects of this invention are: ① This invention first discovered that knocking out NCAM2 in HEK-293T cells can inhibit SVA replication, which can be used as a target for preparing drugs that inhibit SVA replication; ② This invention provides an sgRNA that targets the NCAM2 gene. The sgRNA can specifically target the NCAM2 gene, and combined with CRISPR-Cas9 technology, it can achieve the knockout of the NCAM2 gene in host cells with accurate targeting and high knockout efficiency; ③ This invention provides a method for transfecting the sgRNA into host cells using CRISPR-Cas9 technology to construct an NCAM2 gene knockout cell line. By knocking out the NCAM2 gene, a cell line with an SVA resistance phenotype is obtained. SVA replication in the cell line is significantly inhibited, and viral nucleic acid and titer are significantly reduced. It can significantly inhibit SVA replication, providing research tools and materials for further research on the molecular mechanism of NCAM2 gene regulating pathogenic microorganism replication in cells. It can also be used for animal breeding against SVA, providing some theoretical basis for future prevention or inhibition of SVA infection. Attached Figure Description
[0034] Figure 1 Schematic diagram of sgRNA targeting the NCAM2 genomic region;
[0035] Figure 2 Screening results of NCAM2 gene knockout cell lines;
[0036] Figure 3 Western blot analysis was performed on NCAM2 protein expression in NCAM2 gene knockout cell lines and VP2 protein expression after SVA infection.
[0037] Figure 4 Results of SVAmRNA level detection in NCAM2 gene knockout cell lines infected with SVA;
[0038] Figure 5 Viral titer detection results of NCAM2 gene knockout cell lines infected with SVA;
[0039] Figure 6 Fluorescence detection results of the virus after NCAM2 gene knockout cell lines were infected with SVA. Detailed Implementation
[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the various embodiments of this invention will be described in detail below with reference to the accompanying drawings. However, those skilled in the art will understand that many technical details have been provided in the embodiments of this invention to facilitate a better understanding of this application. However, the technical solutions claimed in this application can be implemented even without these technical details and various variations and modifications based on the following embodiments.
[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent companies.
[0042] The cells, plasmids and viruses, HEK-293T cells and plasmids involved in the following examples were provided by the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences; the SVA-CH-FJ-2017 strain was preserved by the National Foot-and-Mouth Disease Reference Laboratory of the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences.
[0043] The NCAM2 gene sequence described in the following examples is shown in SEQ ID NO.10, and the protein sequence is shown in SEQ ID NO.9.
[0044] Example 1: Construction of NCAM2 gene knockout cell lines
[0045] 1. Primer design and synthesis
[0046] Two sets of sgRNAs specifically targeting NCAM2 were designed and screened based on the NCAM2 gene sequence, and the upstream and downstream primer sequences for these sgRNAs were synthesized. A schematic diagram of the chromatogram of sgRNAs targeting the NCAM2 genomic region is shown below. Figure 1 As shown.
[0047] Target sequence:
[0048] NCAM2-sgRNA1: acagggtagtagtgcaaa (shown in SEQ ID NO.1);
[0049] NCAM2-sgRNA2: ggtgaacctgaaagtatagat (shown in SEQ ID NO. 2).
[0050] Upstream and downstream primer sequences:
[0051] NCAM2-sgRNA1-F: caccgacagggtagtagtgcaaa (shown in SEQ ID NO.3),
[0052] NCAM2-sgRNA1-R: aaacttgcactactaccctctgtc (shown in SEQ ID NO.4);
[0053] NCAM2-sgRNA2-F: caccggtgaacctgaaagtatagat (shown in SEQ ID NO.5),
[0054] NCAM2-sgRNA2-R: aaacatctatactttcaggttcacc (shown in SEQ ID NO. 6).
[0055] 2. Plasmid construction
[0056] Obtaining double-stranded sgRNA-oligo: The synthesized sgRNA-oligo was diluted to 100 μmol / L to prepare a 10 μL reaction mixture: 4.5 μL of upstream primer; 4.5 μL of downstream primer; 1 μL of 10×LA PCR Buffer. The mixture was gently mixed. Annealing program: 95℃, 10 min.
[0057] Enzyme digestion of the PX459 vector plasmid: The PX459 vector was digested with BbsI restriction endonuclease. The 20 μL digestion system was as follows: PX459 vector, 5 μL; BbsI, 1 μL; 10× Buffer, 2 μL; ddH2O, 12 μL. The digestion was carried out at 37℃ for 2 h. Afterwards, nucleic acid electrophoresis was performed, and the linearized fragment of the PX459 vector containing sticky ends was purified and recovered using an Omega DNA purification and recovery kit.
[0058] Construction of the PX459-sgRNA recombinant plasmid: The purified and recovered linearized PX459 fragment was ligated with double-stranded sgRNA-oligo. The reaction system was as follows: T4 Ligase, 1 μL; 10×T4 Ligase Buffer, 1 μL; PX459 digested and purified fragment, 1 μL; double-stranded sgRNA-oligo, 7 μL. Ligation was performed overnight at 16℃. The ligation product was transformed into DH5α Escherichia coli competent cells for cloning and amplification of the recombinant plasmid.
[0059] Single colonies were selected, and omega plasmids were extracted and sequenced using an omega plasmid extraction kit. The sequencing results are as follows: Figure 1 As shown, the sequence obtained from the constructed plasmid using TRC universal primers was compared with the original vector, and the results were consistent with expectations. This indicates that the plasmids PX459-NCAM2-sgRNA1 and PX459-NCAM2-sgRNA2 expressing sgRNA were successfully constructed.
[0060] 3. Construction of NCAM2 gene knockout HEK-293T cell line
[0061] HEK-293T cells were resuscitated in T25 cell culture flasks. After 2-3 passages, when the cells were stable and in good condition, they were digested and seeded into 6-well plates. At a cell density of 70%-80%, the successfully constructed recombinant plasmid was transfected into HEK-293T cells. 24 hours later, the cells were treated with puromycin at a concentration of 2 μg / mL for 2-3 days to select positive cells. Subsequently, 100 positive cells were seeded into 96-well plates to obtain single-cell clones.
[0062] (1) Extraction of cellular DNA and detection of gene targeting efficiency:
[0063] Following the instructions of the micro DNA extraction kit, the genome of a single cell clone was extracted, and the NCAM2 gene fragment containing the target site region was further amplified using DNA check primers (NCAM2-check-sgRNA-F: TTTACTTTAAGTTTTGTAAGA, NCAM2-check-sgRNA AR: AATAAACCACTATTCTCTAT).
[0064] The results are as follows Figure 2 As shown, using wild-type HEK-293T cells as a control, all single-cell clones were amplified to a fragment size of approximately 600 bp, consistent with the expected fragment size. The nucleic acids at the target location were then gel-cleaved, purified, and recovered before sequencing. The sequencing map is shown below. Figure 5 As shown in the sequencing peak diagram, the presence of base deletions indicates that effective gene editing occurred in the amplified fragment.
[0065] (2) Verification of NCAM2-KOs by Western blotting
[0066] HEK-293T cells (wild-type cells) without gene editing were used as a negative control. Wild-type (WT) cells and two knockout candidate cell lines (KO1 and KO2, respectively) were cultured to 70%-80% confluence. A suitable amount of 1×SDS loading buffer was added and the cells were thoroughly lysed. After complete cell detachment, the cells were transferred to EP tubes and labeled. The tubes were then incubated in a metal bath for 15 minutes to denature the cells. After centrifugation, the supernatant was used for SDS-PAGE. Following electrophoresis, the cells were transferred to an NC membrane using a wet transfer method. The membrane was blocked with 5% skim milk powder. Antigen-antibody complex analysis was performed using purchased rabbit NCAM2 antibody as the primary antibody and goat anti-rabbit IgG (IgG-HRP) as the secondary antibody to validate the protein expression level of the NCAM2 gene knockout HEK-293T monoclonal cell line.
[0067] Experimental results are as follows Figure 3 As shown, WT detected a clear NCAM2 protein band, while no NCAM2 protein band was detected in either of the two candidate cell lines. This indicates that the NCAM2 gene was successfully targeted in the two candidate cell lines, KO1 and KO2. This demonstrates that the complete knockout of the NCAM2 gene in the host cell was achieved using the sgRNA provided by this invention and the CRISPR-Cas9 technology.
[0068] Example 2: Effect of NCAM2-KO cells on SVA replication
[0069] 1. Cellular infection
[0070] Wild-type HEK-293T cells (WT) and NCAM2 gene knockout HEK-293T cells (NCAM2-KO1, NCAM2-KO2) were seeded into 12-well plates. When the cell density reached 70%–80%, SVA (MOI=1) was seeded. Samples were collected 12 hours after infection, and the replication status of Seneca virus was detected by Western blot, RT-qPCR, TCID50 and other methods.
[0071] 2. Western blot for protein immunoblotting
[0072] Western blot analysis was performed to detect NCAM2 protein expression in NCAM2 gene knockout cell lines and VP2 protein expression after SVA infection.
[0073] The results are as follows Figure 3 As shown, compared with wild-type HEK-293T cells (WT), NCAM2 gene knockout HEK-293T cells (NCAM2-KO1, NCAM2-KO2) showed no expression of NCAM2 protein, indicating that the NCAM2 gene knockout HEK-293T cells were successfully constructed. After infection with SVA, compared with wild-type HEK-293T cells (WT), the expression level of SVAVP2 protein in NCAM2 gene knockout HEK-293T cells (NCAM2-KO1, NCAM2-KO2) was significantly reduced, indicating that NCAM2 gene knockout significantly inhibited SVA replication.
[0074] 3. Real-time quantitative PCR
[0075] Wild-type HEK-293T cells (WT) and NCAM2 gene knockout HEK-293T cells (NCAM2-KO1, NCAM2-KO2) were collected after SVA infection. Total RNA was extracted according to the Trizol lysis method instructions. After the concentration was determined, cDNA was obtained by reverse transcription using a reverse transcription kit. A 20 μL premixed reaction system was prepared using an Mx3005P-QPCR system (Agilent Technologies, USA) and 10 μL of TBGreen II Premix Ex Taq, 0.5 μL each of forward and reverse primers (SVA-3D-F: AGAATTTTGGAAGCCATCTCT, SVA-3D-R: GAGCCAACATAGARACAGATTGC), 1 μL of cDNA, and 8 μL of ddH2O. The reaction was performed using a 40-cycle regimen: 95℃ for 2 min, 95℃ for 10 s, and 60℃ for 30 s (fluorescence collection). GAPDH was used as an internal control gene to quantify the mRNA level of SVA. All experiments were performed in triplicate. -△△CT Methods for analyzing data.
[0076] The results are as follows Figure 4 As shown, compared with wild-type HEK-293T cells (WT), the content of SVA3D mRNA in NCAM2 gene knockout HEK-293T cells (NCAM2-KO1, NCAM2-KO2) was significantly reduced, indicating that NCAM2 gene knockout significantly inhibited SVA replication.
[0077] 4. TCID 50 Measurement
[0078] SVA was used to infect wild-type HEK-293T cells (WT) and NCAM2 gene knockout HEK-293T cells (NCAM2-KO1 and NCAM2-KO2), respectively. The viral fluid was collected after 12 hours, subjected to three freeze-thaw cycles, centrifuged, and the supernatant was collected. The samples were then cultured in serum-free DMEM medium for 10... -2 ~10 -8 Serial dilutions were performed, and each dilution of the virus was seeded into 96-well plates containing a confluent monolayer of IBRS-2 cells, with 8 wells seeded for each dilution at 100 μL / well. The plates were incubated at 37°C in a 5% CO2 cell culture incubator for 3 days. Cytopathic effects (CPE) were observed and recorded during this period, and TCID50 was calculated and analyzed using the Reed-Muench method.
[0079] TCID 50 The measurement results are as follows Figure 5As shown, compared with wild-type HEK-293T cells (WT), the SVA virus titer in NCAM2 gene knockout HEK-293T cells (NCAM2-KO1, NCAM2-KO2) was significantly reduced, indicating that NCAM2 gene knockout significantly inhibited SVA replication.
[0080] 5. Fluorescence detection
[0081] Wild-type HEK-293T cells (WT) and NCAM2 gene knockout HEK-293T cells (NCAM2-KO1 and NCAM2-KO2) were infected with SVA-GFP fluorescent virus. After 12 hours, the fluorescence intensity of the virus was observed under a fluorescence microscope.
[0082] The results are as follows Figure 6 As shown, compared with wild-type HEK-293T cells (WT), the green fluorescence intensity of NCAM2 gene knockout HEK-293T cells (NCAM2-KO1, NCAM2-KO2) was significantly reduced, indicating that NCAM2 gene knockout significantly inhibited SVA replication.
[0083] The above results indicate that inhibiting the expression of the NCAM2 gene can significantly suppress SVA replication, and it can be used as a target for screening drugs that inhibit SVA replication, providing a new strategy for SVA defense.
[0084] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.
Claims
1. Application of NCAM2 gene / protein as a target in screening drugs for the prevention or treatment of microRNAviridae virus infections; the drug targets NCAM2 gene / protein to inhibit or silence the expression of NCAM2 gene / protein.
2. Application of NCAM2 gene / protein expression inhibitors in the preparation of drugs, pharmaceutical compositions or vaccine compositions for the prevention or treatment of microRNAviridae virus infections.
3. The application as described in claim 2, characterized in that, The NCAM2 gene / protein expression inhibitors include small molecule compounds that inhibit NCAM2 gene expression, small interfering RNAs designed to target the NCAM2 gene, or sgRNAs that target and knock out the NCAM2 gene.
4. The application as described in any one of claims 1-3, characterized in that, The virus in question is a Seneca virus.
5. A specific sgRNA targeting and knocking out the NCAM2 gene, characterized in that, The sgRNA targets at least one of the following target sequences: NCAM2-sgRNA1: acagggtagtagtgcaaa; NCAM2-sgRNA2:ggtgaacctgaaagtatagat.
6. The use of the sgRNA as described in claim 5 in the preparation of drugs for the prevention or treatment of microRNA virus infections, or in NC AM2 gene knockout cell lines.
7. The application as described in claim 6, characterized in that, The virus in question is a Seneca virus.
8. Application of NCAM2 gene knockout cell lines in breeding for resistance to Picornaviridae viruses.
9. The application as described in claim 8, characterized in that, The method for constructing NCAM2 gene knockout cells includes the following steps: (1) Prepare the sgRNA that specifically targets the NCAM2 gene as described in claim 5 by adding a CACC sticky end to the 5' end of the forward sequence of the sgRNA fragment and adding an AAAC sticky end to the 5' end of the reverse sequence, as an sgRNA oligonucleotide targeting the NCAM2 gene. (2) Insert the double-stranded fragment prepared in step (1) into the multiple cloning site of the PX459 expression plasmid vector to obtain a recombinant vector that simultaneously expresses the Cas9 protein gene and the targeting sgRNA sequence. (3) Transfect HEK-293T cells with the recombinant vector prepared in step (2), kill negative cells with puromycin, pick single cells, seed and culture them to obtain NCAM2 gene loss-of-function cell line.
10. The application as described in claim 8 or 9, characterized in that, The virus in question is a Seneca virus.