Grouper SIK2 protein and its coding gene in the application of resisting fish nervous necrosis disease

Abstract

The application discloses application of an Epinephelus radiatus SIK2 protein and an encoding gene thereof to fish neural necrosis disease resistance. The open reading frame (ORF) of the SIK2 protein encoding gene is 2796 bp in length, and encodes 931 amino acids, and the amino acid sequence is shown as SEQ ID NO. 1. The application first finds that the Epinephelus radiatus SIK2 protein is a virus response host restriction factor, can significantly inhibit NNV replication in grouper cells, and is favorable to fish viral neural necrosis disease prevention and treatment.

Description

Technical Field

[0001] This invention belongs to the technical field of biotechnology; specifically, it relates to the application of grouper SIK2 protein and its encoding gene in the treatment of fish nerve necrosis. Background Technology

[0002] Grouper ( Epinephelus spp Grouper (Amur grouper) is an important marine economic fish species, widely farmed in coastal areas of my country and Southeast Asia, with its farming scale continuously expanding in recent years. However, viral diseases remain one of the main bottlenecks restricting the sustainable development of grouper farming. Among them, Nervous necrosis virus (NNV), a typical aquatic RNA virus, mainly causes viral nervous necrosis (VNN) in fish, infecting various marine and freshwater fish, including grouper, and is particularly highly pathogenic to larvae and young fish. Grouper (Amur grouper) is an example. Epinephelus coioides It has been proven to be a highly susceptible host for NNV, and its repeated outbreaks during the breeding process have caused serious economic losses to the industry.

[0003] In lower vertebrates such as grouper, the innate immune system plays a crucial role in antiviral defense. Therefore, elucidating the innate immune mechanism against NNV in grouper and identifying key antiviral genes has become an urgent need in the field of aquatic disease control. In these animals, the innate immune system is the first line of defense against pathogen invasion. The core of innate immunity is the recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs), which then activates downstream immune signaling cascades. Among these, the signaling pathway mediated by retinoic acid-induced gene I (RIG-I)-like receptors (RLRs) plays a key role in host defense against RNA viruses. Within the RLR pathway, interferon regulatory factors (IRFs) and nuclear factor κB (NF-κB), as key transcription factors, are activated by upstream PRRs and translocate into the nucleus, inducing the expression of interferons (IFNs) and pro-inflammatory cytokines, thereby establishing an antiviral immune response.

[0004] Furthermore, mounting evidence suggests that liquid-liquid phase separation (LLPS) plays a crucial role in the regulation of innate immunity. Proteins containing intrinsically disordered regions (IDRs) can form biomolecular condensates through LLPS, promoting key immune processes such as pattern recognition receptor activation and antiviral signaling complex assembly.

[0005] Salt-inducible kinases (SIKs) belong to the AMP-activated protein kinase (AMPK) family, comprising three subtypes: SIK1, SIK2, and SIK3. SIK2 is highly expressed in adipose tissue and plays a crucial role in regulating metabolic homeostasis. To date, research on SIK2 has primarily focused on mammals, covering aspects such as metabolic homeostasis, autophagy, and tumor-related signaling pathways. In bony fish, studies have been conducted on large yellow croaker (…). Larimichthys crocea Studies have shown that SIK2 can reduce inflammatory responses by regulating hepatic glucose and lipid metabolism, but the role of SIK2 in viral infection, especially in antiviral immunity in bony fish, has not been reported, and its potential antiviral function and molecular mechanism remain unclear.

[0006] Therefore, identifying and demonstrating key host regulatory factors against NNV in grouper and elucidating their mechanisms of action is of great significance for developing novel prevention and control strategies and immune enhancers for fish viral diseases. Summary of the Invention

[0007] The present invention aims to provide a SIK2 protein from the grouper, its encoding gene, and a recombinant vector containing the gene. The grouper SIK2 protein is a host-restricting factor in response to a virus, capable of synergistically inhibiting the replication of fish neuronecrosis virus by activating TBK1-related signals and NF-κB signals, and its antiviral activity depends on liquid-liquid phase separation characteristics.

[0008] The present invention also aims to provide the application of the SIK2 protein of the oblique grouper or its encoding gene or the recombinant vector in the treatment of NNV and the resulting viral neuronecrosis in fish.

[0009] The first objective of this invention can be achieved through the following technical solution:

[0010] A SIK2 protein from the grouper, named EcSIK2, is derived from the grouper. The open reading frame (ORF) of the EcSIK2 encoding gene is 2796 bp in length and encodes 931 amino acids, the amino acid sequence of which is shown in SEQ ID NO.1.

[0011] Structural predictions indicate that EcSIK2 contains an N-terminal serine / threonine kinase domain (S_TKc, located at amino acid residues 21-272) and two intrinsically disordered regions (IDRs) at the C-terminus, named the PL domain (located at amino acid residues 391-683) and the PG domain (located at amino acid residue 743 to the C-terminus).

[0012] The present invention also provides the gene encoding the SIK2 protein, hereinafter referred to as Ecsik2, whose nucleotide sequence is shown in SEQ ID NO.2.

[0013] The present invention also provides a recombinant vector containing the coding gene; the recombinant vector is a eukaryotic expression vector. Preferably, the eukaryotic expression vector is pEGFP-N3.

[0014] This invention reveals that the SIK2 protein in the grouper is a host-restricting factor in viral response, enhancing the fish's resistance to NNV. Specifically, overexpression of the EcSIK2 plasmid in fish cells significantly inhibits NNV replication and reduces the expression levels of viral capsid protein (CP) and RNA-dependent RNA polymerase (RdRp); conversely, knockdown of EcSIK2 expression promotes NNV replication.

[0015] The EcSIK2 enhances fish's resistance to NNV through the following mechanisms: Overexpression of the EcSIK2 plasmid in fish cells did not significantly alter the transcriptional levels of pattern recognition receptor genes (mda5, znfx1) or adaptor genes (mavs), but selectively upregulated traf3 expression. The mRNA levels of key signaling cytokines (tbk1, irf3, irf7, nf-κb), interferons (ifnc, ifnh), interferon-stimulated genes (ifit1, isg15), and pro-inflammatory cytokines (tnfα, il-1β) were all significantly increased after EcSIK2 plasmid overexpression. Consistent with this, knockdown of EcSIK2 suppressed the expression of irf3, ifnc, and tnfα, indicating that EcSIK2 is a positive regulator of antiviral immunity.

[0016] The EcSIK2 also enhances fish's resistance to NNV through the following mechanisms: EcSIK2 physically interacts with TBK1, IRF3, IRF7 and NF-κB, enhancing the phosphorylation of TBK1 and IRF3, while activating the promoter activity of the nf-κb promoter and interferon-related genes (ifnc, ifit1), thereby coordinating the activation of two antiviral signaling pathways: (i) the EcSIK2-NF-κB-pro-inflammatory cytokine axis; (ii) the EcSIK2-TBK1-IRF3-interferon axis.

[0017] Specifically, the C-terminal PG domain of EcSIK2 mediates liquid-liquid phase separation, and EcSIK2 forms liquid-like cytoplasmic aggregates in cells. These aggregates can be dissolved by 3% 1,6-hexanediol and exhibit rapid recovery after fluorescent bleaching and dynamic fusion / division. The phase separation capability mediated by the PG domain is essential for the complete antiviral activity of EcSIK2. Deletion of the PG domain leads to reduced aggregate formation and a significant decrease in antiviral activity.

[0018] The second objective of this invention can be achieved through the following technical solution:

[0019] The application of the SIK2 protein of the grouper or its encoding gene or the above-mentioned recombinant vector in the fight against NNV is specifically in the preparation of anti-NNV drugs and in the breeding of anti-NNV.

[0020] Furthermore, the present invention provides the application of the *Sinocyclocheilus scoparia* SIK2 protein or its encoding gene, or the recombinant vector, in resistance to NNV-induced viral neuronecrosis in fish. Specifically, the application of the *Sinocyclocheilus scoparia* SIK2 protein or its encoding gene, or the aforementioned recombinant vector, in the preparation of drugs against fish viral neuronecrosis and in the breeding of fish resistant to viral neuronecrosis.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) This invention first discovered that the SIK2 protein EcSIK2 of the grouper is a host restriction factor for viral response, which can significantly inhibit the replication of NNV in grouper cells, expand the functional framework of SIK2, and extend it from metabolic regulation to the field of antiviral host defense.

[0023] (2) This invention reveals the molecular mechanism by which EcSIK2 exerts its effects by coordinating two anti-NNV virus signaling pathways: EcSIK2 interacts with TBK1, IRF3, IRF7 and NF-κB, enhances the phosphorylation of TBK1 and IRF3, and activates the NF-κB-pro-inflammatory cytokine axis and the TBK1-IRF3-interferon axis, thereby achieving synergistic activation of inflammatory response and interferon-related antiviral response.

[0024] (3) This invention is the first to discover that EcSIK2 has liquid-liquid phase separation characteristics, its C-terminal PG domain mediates condensate formation, and the phase separation ability is directly related to antiviral activity. The functional coupling between the kinase domain and the phase separation-related region is the basis of the complete antiviral activity of EcSIK2, providing a new perspective for understanding the spatial organization and regulation of antiviral signals.

[0025] (4) The EcSIK2 of the present invention significantly inhibits the replication of NNV in grouper cells and can be developed into a drug for fish to resist NNV or a breeding target for anti-NNV genes. This is beneficial to the prevention and treatment of viral nerve necrosis in grouper and other perch-like fish and is of great significance to the healthy development of marine fish aquaculture. Attached Figure Description

[0026] Figure 1The expression profile of EcSIK2 and its inhibitory effect on OGNNV replication are shown. Wherein: A represents the relative mRNA expression level of EcSIK2 in different tissues of *Sinocyclocheilus fasciatus*; BC represents the relative mRNA expression levels of Ecsik2 (B) and cp (C) in GS cells within 48 h after OGNNV infection; D represents the relative mRNA level of Ecsik2 in GS cells after transfection with GFP-labeled B1, B2, CP, or RdRp expression plasmids; E represents the relative mRNA level of Ecsik2 in GS cells after stimulation with LMW- or HMW-poly(I:C); F represents the relative mRNA level of Ecsik2 in GS cells after stimulation with rIFNd; GI represents the relative mRNA expression levels of Ecsik2 (G), cp (H), and rdrp (I) in GS cells after overexpression of EcSIK2 plasmid and infection with OGNNV; J represents the expression level of CP protein in GS cells after overexpression of EcSIK2 plasmid and infection with OGNNV. The heatmap on the right shows three independent Western spectroscopy analyses. Quantitative results of blotting experiments; KM represents the relative mRNA expression levels of Ecsik2 (K), cp (L), and rdrp (M) in GS cells transfected with siNC or siEcSIK2 and infected with OGNNV. *P < 0.05, **P < 0.01, ***P < 0.001, NS indicates no statistical significance.

[0027] Figure 2 EcSIK2 positively regulates the immune response. Wherein: A and D represent the mRNA expression levels of pattern recognition receptors and adaptor proteins (A), core signal kinases and transcriptional regulators (B), interferons (C), ISGs, and pro-inflammatory cytokines (D) in GS cells overexpressing the EcSIK2 plasmid and infected with OGNNV, respectively; E represents the mRNA expression levels of irf3, ifnc, and tnfα in GS cells transfected with siNC or siEcSIK2 and infected with OGNNV. *P < 0.05, **P < 0.01, ***P < 0.001, NS indicates no statistical significance.

[0028] Figure 3The interactions of EcSIK2 with TBK1, IRF3, IRF7, and NF-κB are shown. Specifically: A represents the results of Co-IP analysis using anti-HA magnetic beads after co-transfection of EcSIK2-HA plasmid and GFP-labeled EV, EcTBK1, EcMAVS, EcMDA5, EcNF-κB, or EcIRF3 / 7 plasmids in HEK 293T cells; B shows the co-localization of the protein with EcSIK2-HA as indicated by immunofluorescence detection, with fluorescence intensity analyzed along the white line to assess co-localization (scale bar: 10 μm); C and D represent the results of Co-IP analysis using anti-GFP magnetic beads after co-transfection of EcIRF3 / 7-myc plasmid and GFP-labeled EV, wild-type EcSIK2, or its mutant plasmids in HEK 293T cells.

[0029] Figure 4 EcSIK2-mediated phosphorylation enhances the host immune response. In the figures: AC represent the promoter activities of nf-κb (A), ifnc (B), and ifit1 (C) after transfection of GS cells with the indicated plasmid, respectively; pGL3-IFNc-mut refers to the ifnc promoter construct with the IRF binding site deleted; DE represents the phosphorylation of EcSIK2 in HEK 293T cells (D) and its effect on TBK1 phosphorylation (E) detected using anti-pan-phosphorylated serine / threonine antibody; FG represents the phosphorylation levels of exogenous (F) and endogenous (G) TBK1 in GS cells overexpressing the EcSIK2 plasmid, detected using anti-p-TBK1 antibody; H represents the expression of phosphorylated IRF3 in HEK 293T cells transfected with the indicated plasmid, detected by immunofluorescence. Red indicates anti-HA or anti-myc, green indicates anti-p-IRF3, and the yellow box area in the combined figure is enlarged in the rightmost column, with a scale bar of 10 μm. **P < 0.01, ***P < 0.001.

[0030] Figure 5 To validate the phase separation properties of EcSIK2. A shows the EcSIK2 phase separation domain predicted using IUPred3; B shows the subcellular localization and phase separation characteristics of EcSIK2 in HEK 293T and GS cells, scale bar 10 μm; C shows the fluorescence images of HEK 293T cells expressing EcSIK2 under normal conditions (Control) and after treatment with 3% 1,6-HD, scale bar 10 μm; D shows the fluidity of EcSIK2 protein as analyzed by FRAP, scale bar 10 μm; E shows the fusion and division of EcSIK2 punctate aggregates, with arrows indicating dynamic fusion and division processes, scale bar 10 μm.

[0031] Figure 6Functional analysis of IDR-mediated antiviral activity. Wherein: A is a schematic diagram of EcSIK2 and its truncated mutants; B shows the phase separation of different EcSIK2 mutants in HEK 293T cells, scale bar 10 μm; CE shows the mRNA expression levels of cp and rdrp (C), CP protein expression level (D), and irf3 and nf-κb mRNA expression levels (E) after transfection of GS cells with EcSIK2 and its mutant plasmids. *P < 0.05, **P < 0.01, ***P < 0.001, NS indicates no statistical significance. Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following embodiments are commercially available.

[0033] The GS cells used in this embodiment of the invention are grouper spleen cell lines derived from the spleen of oblique grouper; the SC1 cell line used was obtained by limiting dilution subcloning of the SSN-1 cell line and was used for virus propagation; the nerve necrosis virus used was isolated from diseased oblique grouper and was the OGNNV strain; all the above materials were preserved in the laboratory of He Jianguo's team at the School of Life Sciences, Sun Yat-sen University.

[0034] Example 1: Cloning, sequence characteristics, and expression pattern analysis of the Ecsik2 gene

[0035] 1. Cloning of the Ecsik2 gene

[0036] Total RNA was extracted from GS cells using the Eastep® Super Total RNA Extraction Kit (Promega), and cDNA first-strand template was prepared by reverse transcription using the PrimeScript II 1st Strand cDNA Synthesis Kit (TAKARA). Based on the nucleotide sequence information of the ecsik2 gene obtained from the transcriptome data, specific primers EcSIK2-ORF-F and EcSIK2-ORF-R (sequences shown in Table 1 below) were designed. PCR amplification was performed using GS cell cDNA as a template to obtain the open reading frame (ORF) of the Ecsik2 gene, as shown in SEQ ID NO.2. The amplified product was detected by agarose gel electrophoresis, recovered from the gel, ligated into pEGFP-N3 and pN3-HA vectors, transformed into E. coli DH5α competent cells, and single colonies were picked for PCR verification and then sequenced for confirmation.

[0037] Sequencing results showed that the ORF of Ecsik2 is 2796 bp in length, encoding 931 amino acids, as shown in SEQ ID NO. 1. Conserved domain prediction using the SMART program (https: / / smart.embl.de / ) revealed that EcSIK2 contains an N-terminal serine / threonine kinase domain (S_TKc, located at amino acid residues 21-272). IUPred3 (https: / / iupred3.elte.hu / ) prediction of the intrinsically disordered region showed that the C-terminal region of EcSIK2 contains two discontinuous IDRs, named the PL domain (located at amino acid residues 391-683) and the PG domain (located from amino acid residue 743 to the C-terminus).

[0038] The amino acid sequence of EcSIK2 is shown below (SEQ ID NO.1):

[0039] MVMADSVQKPLIRGPVRVGFYDIERTLGKGNFAVVKLARHRITKTEVAIKIIDKTQLDAVNLEKIYREVQIMKMLDHPHIIKLYQVMETKNMLYLVTEYAKNGEIFDYLAKHGRLSELEARRKFWQILSAVEYCHNRNIVHRDLKAENLLLDGHMNIKIADFGFGNFFQPGEPLATWCGSPPYAAPEVFEGQQYEGPQLDIWSMGVVLYVLVCGALPFDGPTLPVLRQRVLEGRFRIPYFMTEDCEHLIRRMLVLDPSKRLSVAQIKEHKWMALYAPVQRPVLYQQPLSAEGEAGVGEYSEQVLRLMHSLGIDQHKTIESLQNKSYNHFAAIYYLLVERLKAHRCSFPVEQRLDARQRRPSTIAEQTVVKSTSGSTQVGLLPQGVRLLRSPAVPQASSDAFTFPQTACPLEQNFMVEDVNTPKVNGCLLDPLPPPTVLRKSSTSSPSNMMETSIDEGIETEEPDTEDDPPHMLSAYQTARFGQRRHTLSEVTNQPGPSNQGKLFTLGHNPSMGSVDSDIGYDMGSMHSDFGLLEDPPSLSEVVSANSSAPGVTPTPFLSARPANPAMAVLTSQHRETHNRSPISFREGRRASDTSLTQGLVAFRQHLQNLARTKGILELNKVQMLVEQMGSGEGAAMGPLGPQHHLHNLLEGEASKQQEGLALYQGGPQPPLLSRRQSLETQYLTHRLQANVLANSPASCQLFCKDTRTLEQQLQEHRLNQKRMYLQQSQGMGLQAYFNQMQIAEGSYPTGGPTLDPAASQSSPQGPPMSATHQPQPQQQGSPQASPPFSHPHSLSPLMEPSEGLIYDPYMSHHHYAQHLPPGSHLHHQLHHPQPHDASASSLGFSYPAGCEQQALGPSTEGLHPEHYDFPLDPALLPLPAPGEAGAGAVARILPGPGQSNGLGLPELQGSLLDSEMMETVDSQHGFVLVN*。

[0040] The nucleotide sequence of the EcSIK2 encoding gene, Ecsik2 (ORF region), is shown below (SEQ ID NO.2):

[0041]

[0042] 2. Tissue distribution analysis of EcSIK2

[0043] Nine healthy grouper (average weight 10 g, average body length 8 cm) were selected. Head kidney, fins, gills, intestine, spleen, heart, brain, muscle, eye, and liver tissues were collected and immediately stored in RNAlater after being cut into small pieces. Total RNA was extracted from each tissue, reverse transcribed into cDNA, and the expression of Ecsik2 was detected by quantitative PCR, using β-actin as an internal reference gene and primer Q-SIK2 (see Table 1).

[0044] The results are as follows Figure 1 As shown in Figure A, the transcript abundance of EcSIK2 varies significantly among different tissues of the grouper, with the highest expression level in the eye, approximately 30 times that in the head kidney; while the expression level is lower in the intestine, gills, and fins.

[0045] 3. Effects of OGNNV infection on EcSIK2 expression

[0046] GS cells were seeded into 6-well plates and cultured for 12 h until 80% confluence. The cells were then infected with OGNNV at a multiplicity of infection (MOI) of 2. After adsorption at 28°C for 2 h, the inoculum was discarded, and fresh DMEM medium containing 2% FBS was added. Cells were collected at 12 h, 24 h, 36 h, and 48 h post-infection. Total RNA was extracted and reverse transcribed into cDNA. The expression levels of Ecsik2 and cp were detected by quantitative PCR using primers Q-SIK2 and Q-CP (see Table 1).

[0047] The results are as follows Figure 1 As shown in BC, during OGNNV infection, the mRNA level of cp gradually increased from 12 h to 24 h and peaked at 24 hpi; while the transcriptional level of Ecsik2 decreased from 12 to 24 h post-infection and then rebounded from 36 to 48 h, indicating that the expression of Ecsik2 is dynamically related to viral replication dynamics.

[0048] 4. Effects of different stimuli on EcSIK2 expression

[0049] (1) Effect of viral protein overexpression: GS cells were seeded in 6-well plates and cultured for 12 h. Then, they were transfected with OGNNV viral protein expression plasmids (2 μg per well) tagged with GFP, including B1, B2, CP, and RdRp. Cells were collected and RNA was extracted using primer Q-SIK2 (see Table 1). Results are as follows: Figure 1 As shown in Figure D, the RdRp plasmid overexpressing OGNNV significantly induced Ecsik2 expression, while B1, B2, and CP had no significant effect on Ecsik2 expression.

[0050] (2) Effect of interferon inducer poly(I:C) stimulation: GS cells were seeded in 6-well plates and cultured for 12 h. The cells were then treated with 5 μg / mL of low molecular weight poly(I:C) (LMW) or high molecular weight poly(I:C) (HMW). Cells were collected at specified time points to detect Ecsik2 expression using primer Q-SIK2. The results are as follows: Figure 1 As shown in E, both low-molecular-weight and high-molecular-weight poly(I:C) stimulation inhibited Ecsik2 expression.

[0051] (3) Effect of recombinant IFNd treatment: GS cells were seeded in 6-well plates and cultured for 12 h. The cells were then treated with recombinant IFNd (rIFNd) at a final concentration of 3 μg / mL. Cells were collected at 1 h, 3 h, 6 h, and 12 h to detect Ecsik2 expression. The results are as follows: Figure 1 As shown in F, rIFNd treatment did not affect the mRNA expression level of Ecsik2 within 1–12 h.

[0052] The above results indicate that Ecsik2 is a virus-induced gene rather than an interferon-stimulated gene, meaning that the expression of Ecsik2 is induced by the virus.

[0053] Example 2: Functional Verification of EcSIK2 in Suppressing OGNNV Replication

[0054] 1. Effect of EcSIK2 overexpression on OGNNV replication

[0055] GS cells were seeded in 6-well plates and cultured for 12 h until 80% confluence. Then, 2 μg of the Ecsik2 overexpression plasmid was transfected, along with an equal amount of empty vector (EV) as a control. 24 h after transfection, GS cells were infected with OGNNV at MOI = 2. Cells were collected at specified time points, and total RNA was extracted for quantitative PCR to detect the expression levels of viral genes cp, rdrp, and Ecsik2. Primers Q-SIK2, Q-CP, and Q-RdRp were used (see Table 1). The expression level of viral CP protein was also detected by Western blotting.

[0056] Quantitative PCR results as follows Figure 1 As shown in the GI, compared with the control group, the mRNA expression levels of cp and rdrp were significantly reduced in GS cells overexpressing the EcSIK2 plasmid. Western blotting results are shown below. Figure 1 As shown in Figure J, the expression level of CP protein in GS cells overexpressing the EcSIK2 plasmid was significantly reduced at 24 and 48 hpi. The heatmap on the right shows the results of three independent experiments.

[0057] 2. The impact of EcSIK2 knockdown on OGNNV replication

[0058] Three siRNAs targeting the mRNA transcribed from Ecsik2 (siEcSIK2-1, siEcSIK2-2, and siEcSIK2-3, sequences shown in Table 1) were designed and synthesized, and siEcSIK2 was prepared by mixing them in equal volumes. GS cells were seeded in 12-well plates and cultured for 12 h. Then, siNC (negative control siRNA) or siEcSIK2 was transfected using Lipo8000 transfection reagent (Beyotime) to a final concentration of 50 nM. Twenty-four h after transfection, GS cells were infected with OGNNV at an MOI of 2. Cells were collected at specified time points, and the expression levels of Ecsik2, cp, and rdrp were detected by quantitative PCR using primers Q-SIK2, Q-CP, and Q-RdRp.

[0059] The results are as follows Figure 1 As shown in KM, compared with the siNC control group, siEcSIK2 transfection significantly knocked down Ecsik2 expression, while the mRNA expression levels of cp and rdrp significantly increased.

[0060] The above-mentioned gain-of-function and loss-of-function experiments together demonstrate that EcSIK2 can significantly inhibit the replication of OGNNV in GS cells.

[0061] Example 3: Study on the regulation of immune response and molecular mechanism of EcSIK2

[0062] 1. Effects of EcSIK2 on the expression of immune-related genes

[0063] GS cells were seeded in 6-well plates and transfected with EcSIK2 overexpression plasmid and empty vector, respectively. 24 h after transfection, cells were infected with OGNNV at an MOI of 2. 48 hours post-infection... RNA was extracted from cells, and the expression levels of a series of immune-related genes were detected by quantitative PCR, including: pattern recognition receptors and adaptor proteins (mda5, znfx1, mavs, traf3), core signal kinases and transcription regulators (tbk1, irf3, irf7, nf-κb), interferons (ifnc, ifnh), ISGs and pro-inflammatory cytokines (ifit1, isg15, mx1, tnfα, il-1β, il-8), using primers Q-MDA5, Q-ZNFX1, Q-MAVS, Q-TRAF3, Q-TBK1, Q-IRF3, Q-IRF7, Q-NF-κB, Q-IFNc, Q-IFNh, Q-IFIT1, Q-ISG15, Q-MX1, Q-TNFα, Q-IL-1β, and Q-IL-8 (see Table 1).

[0064] The results are as follows Figure 2 As shown in AD, overexpression of the EcSIK2 plasmid did not alter the expression of pattern recognition receptors (mda5, znfx1) or adaptor genes (mavs), but selectively enhanced the transcription of traf3; the mRNA expression levels of core signal kinases and transcriptional regulators (tbk1, irf3, irf7, nf-κb), interferons (ifnc, ifnh), some ISGs and pro-inflammatory cytokines (ifit1, isg15, tnfα, il-1β) were all significantly upregulated.

[0065] In the knockdown experiment, GS cells were transfected with siNC or siEcSIK2 and then infected with OGNNV. The expression of irf3, ifnc, and tnfα was detected using primers Q-IRF3, Q-IFNc, and Q-TNFα (see Table 1). The results are as follows: Figure 2 As shown in Figure E, siRNA-mediated Ecsik2 knockdown attenuated OGNNV-induced expression of irf3, ifnc, and tnfα. These results establish EcSIK2 as a positive regulator of antiviral immunity.

[0066] 2. Analysis of the interaction between EcSIK2 and immune signaling proteins

[0067] (1) Co-immunoprecipitation (Co-IP) experiment: HEK 293T cells were seeded in 6-well plates and co-transfected with EcSIK2-HA plasmid and GFP-labeled EV, EcTBK1, EcMAVS, EcMDA5, EcNF-κB or EcIRF3 / 7 plasmids. 48 h after transfection, cells were lysed with cell lysis buffer (Beyotime, with added protease inhibitor mixture) at 4°C for 30 min, and centrifuged at 12000 × g for 5 min to collect the supernatant. Immunoprecipitation was performed by incubation with anti-HA magnetic beads (ThermoFisher) for 1 h. After washing the magnetic beads 3 times with TBST, SDS-PAGE loading buffer was added and the cells were boiled for 10 min for Western blotting.

[0068] The results are as follows Figure 3 As shown in Figure A, EcSIK2 exhibits physical interactions with TBK1, IRF3, IRF7, and NF-κB, while no interactions were detected with MAVS or MDA5.

[0069] (2) Immunofluorescence colocalization analysis: GS cells or HEK 293T cells were seeded on 14 mm coverslips, transfected with the indicated plasmids, and cultured for 24 h. They were fixed with 4% paraformaldehyde (Biosharp) for 10 min, permeabilized with 0.2% Triton X-100 (Sangon Biotech) for 10 min, and blocked with 5% goat serum (BOSTER) at room temperature for 1 h. Primary antibody was added and incubated at 4℃ for 12 h. After washing three times with TBST, the corresponding fluorescent secondary antibody (Alexa Fluor 594 or 488, abcam) was added and incubated at room temperature for 1 h. The nuclei were stained with Hoechst 33342 (Beyotime). Observation was performed using a Leica TCS SP8 STED 3X microscope.

[0070] The results are as follows Figure 3 As shown in Figure B, EcSIK2 co-localizes with TBK1, IRF3, IRF7 and NF-κB within the cell, further supporting the physical association between them.

[0071] (3) Interaction analysis of kinase-deficient mutants: A kinase domain-deficient mutant (ΔS_TKc) and a kinase domain-only mutant (S_TKc) of EcSIK2 were constructed. These mutants were co-transfected with EcIRF3 / 7-myc plasmid and GFP-labeled EV, wild-type EcSIK2, S_TKc, or ΔS_TKc mutant plasmids in HEK 293T cells. Co-immunoprecipitation (Co-IP) was performed using anti-GFP magnetic beads (Smart-lifeSciences). Results are shown below. Figure 3 As shown in CD, the kinase-deficient ΔS_TKc mutant still retains its interaction with IRF3 / 7, indicating that the binding of EcSIK2 to IRF3 / 7 is independent of kinase activity.

[0072] 3. Regulation of promoter activity by EcSIK2

[0073] GS cells were seeded in 12-well plates and co-transfected with 1 μg of empty vector (EV) or EcSIK2-HA expression vector, 200 ng of firefly luciferase reporter vector (pGL3-IFNc or ​​pGL3-IFNc-mut (with the IRF binding site "CAGAAACTGAAACT" deleted), pGL3-NF-κB, pGL3-IFIT1), and 20 ng of internal control Renida luciferase vector pRL-CMV. Cells were collected 48 h after transfection, and the activities of firefly and Renida luciferase were measured using a dual-luciferase reporter gene assay kit (Promega).

[0074] The results are as follows Figure 4As shown in AC, EcSIK2 can significantly activate the promoter activity of nf-κb, ifnc, and ifit1. Deletion of the IRF binding site (pGL3-IFNc-mut) in the ifnc promoter can significantly weaken the activation mediated by EcSIK2, reducing it by about 2.19 times compared with the wild-type promoter.

[0075] 4. Phosphorylation analysis of EcSIK2

[0076] (1) Phosphorylation of EcSIK2: EcSIK2-HA expression plasmid was transfected into HEK 293T cells. 48 h after transfection, immunoprecipitation was performed using anti-HA magnetic beads, followed by Western blotting using an anti-pan-phosphorylated serine / threonine antibody (ABclonal). Results are as follows: Figure 4 As shown in D, EcSIK2 can undergo serine / threonine phosphorylation.

[0077] (2) Effect of EcSIK2 on TBK1 phosphorylation: EcSIK2-HA and TBK1-Flag expression plasmids were co-transfected into HEK 293T cells. 48 h after transfection, TBK1 was immunoprecipitated using anti-Flag magnetic beads (Smart-life Sciences), and the phosphorylation level of TBK1 was detected using anti-pan-phosphorylated serine / threonine antibody. The results are as follows: Figure 4 As shown in E, EcSIK2 can enhance the phosphorylation of TBK1.

[0078] After overexpressing the EcSIK2 plasmid in GS cells, the cells were treated with 5 μg / mL LMW-poly(I:C) for 6 h. Cells were then collected and Western blotting was performed using an anti-p-TBK1 antibody (anti-p-TBK1, Cell Signaling). Results are as follows: Figure 4 As shown in FG, overexpression of the EcSIK2 plasmid can enhance the phosphorylation levels of both exogenous and endogenous TBK1.

[0079] (3) Effect of EcSIK2 on IRF3 phosphorylation: The plasmid shown was transfected into HEK 293T cells, and the expression of phosphorylated IRF3 (p-IRF3) was detected by immunofluorescence. The results are as follows: Figure 4 As shown in H, EcSIK2 can drive the phosphorylation of IRF3.

[0080] The above results preliminarily establish that EcSIK2, as a kinase, activates the host immune response by targeting interferon and pro-inflammatory factors, thereby jointly restricting OGNNV replication; and reveal two parallel pathways: (i) the EcSIK2-NF-κB-pro-inflammatory cytokine axis; and (ii) the EcSIK2-TBK1-IRF3-IFN axis.

[0081] Example 4: Verification and Functional Analysis of EcSIK2 Phase Separation Characteristics

[0082] 1. Bioinformatics prediction of phase separation characteristics of EcSIK2

[0083] The intrinsic disordered regions of EcSIK2 were predicted using the online tool IUPred3 (https: / / iupred3.elte.hu / ). The results are as follows: Figure 5 As shown in Figure A, the C-terminal region of EcSIK2 contains IDRs, and two discontinuous IDRs were precisely identified: the PL domain (residues 391-683) and the PG domain (residues 743 to the C-terminus).

[0084] 2. Observations on the formation of EcSIK2 condensates

[0085] HEK 293T or GS cells were seeded onto 14 mm coverslips, transfected with the EcSIK2-GFP expression plasmid, cultured for 24 h, fixed with 4% paraformaldehyde, stained with Hoechst 33342, and observed using a Leica TCS SP8 STED 3X microscope. Results are as follows: Figure 5 As shown in B, EcSIK2 can form liquid cytoplasmic aggregates in both HEK 293T cells and GS cells, indicating that it has similar phase separation characteristics in mammalian and fish cells.

[0086] 3. 1,6-Hexanediol (1,6-HD) treatment experiment

[0087] HEK 293T cells were seeded in glass-bottomed culture dishes and transfected with the EcSIK2-GFP plasmid. 1,6-HD (Sigma-Aldrich) was dissolved in complete cell culture medium to a final concentration of 3% (w / v), filtered through a 0.22 μm filter for sterilization, and preheated to 37°C. The original culture medium was gently aspirated, and an equal volume of preheated drug-containing medium was added for live-cell fluorescence imaging. Results are shown below. Figure 5 As shown in Figure C, after treatment with 3% 1,6-HD, the EcSIK2 aggregates dissolved, and the fluorescence signal became diffuse, indicating that the formation of aggregates depends on hydrophobic interactions.

[0088] 4. Fluorescence Recovery After Bleaching (FRAP) Analysis

[0089] HEK 293T cells were seeded in glass-bottomed culture dishes and transfected with the EcSIK2-GFP plasmid. FRAP experiments were performed using a Leica TCS SP8STED 3X microscope. The GFP signal was photobleached using a 488 nm laser beam, and the fluorescence recovery at specified time points after bleaching was recorded. Results are as follows: Figure 5 As shown in Figure D, the EcSIK2 condensate recovered rapidly after photobleaching, indicating that it has fluidity, which is consistent with the characteristics of liquid-liquid phase separation.

[0090] 5. Observation of fusion and splitting of aggregates

[0091] HEK 293T cells were seeded in glass-bottomed culture dishes, transfected with the EcSIK2-GFP plasmid, and observed under a time-lapse fluorescence microscope. Results are as follows: Figure 5 As shown in Figure E, splitting events (0–12 s) and fusion events (12–17 s) of the EcSIK2 condensate were observed, verifying the dynamic splitting / fusion characteristics of biomolecular phase separation.

[0092] 6. Phase segregation and antiviral function analysis of domain-truncated mutants

[0093] A series of domain-truncated mutants of EcSIK2 were constructed using PCR, including: S_TKc (containing only the kinase domain), ΔS_TKc (lacking the kinase domain), PG (containing only the PG domain), and ΔPG (lacking the PG domain). PCR amplification was performed using the EcSIK2-EGFP recombinant plasmid as a template and specific primers. All mutants were validated by sequencing.

[0094] (1) Phase separation ability analysis: HEK 293T cells were transfected with plasmids of each mutant, and aggregate formation was observed under a fluorescence microscope 24 h later. The results are as follows: Figure 6 As shown in AB, the PG domain determines the phase separation ability of EcSIK2, and the ΔPG mutant forms significantly fewer aggregates.

[0095] (2) Antiviral function analysis: GS cells were seeded in 6-well plates and transfected with wild-type EcSIK2 and various mutant plasmids, respectively. 24 h after transfection, cells were infected with OGNNV with MOI = 2. Cells were collected 48 h after infection. The mRNA levels of cp and rdrp, as well as the expression of irf3 and nf-κb, were detected by quantitative PCR. The CP protein level was detected by Western blotting. Primers Q-CP, Q-RdRp, Q-IRF3, and Q-NF-κB were used (see Table 1).

[0096] The results are as follows Figure 6As shown in CE, deletion of the PG domain (ΔPG) significantly increased viral mRNA and protein levels, and other mutants also exhibited impaired antiviral activity; simultaneously, the irf3 and nf-κb induction abilities of these mutants were also weakened. These results indicate that both the kinase domain and the PG domain are essential for the complete antiviral activity of EcSIK2.

[0097] Table 1 Primers and siRNA sequences used in this invention

[0098] The above embodiments are only used to illustrate the present invention, and the scope of protection of the present invention is not limited to the above embodiments. Those skilled in the art can achieve the purpose of the present invention based on the above disclosure. Any improvements and modifications made based on the concept of the present invention fall within the scope of protection of the present invention, and the specific scope of protection is determined by the claims.

Claims

1. The use of the SIK2 protein of the grouper, or the encoding gene of the SIK2 protein, or a recombinant vector containing the encoding gene, in the preparation of an anti-neural necrosis virus drug.

2. Application of SIK2 protein of grouper or the encoding gene of said SIK2 protein or a recombinant vector containing said encoding gene in breeding against nerve necrosis virus.

3. The use of SIK2 protein of the grouper, or the encoding gene of said SIK2 protein, or a recombinant vector containing said encoding gene, in the preparation of a drug for treating viral nerve necrosis in fish.

4. Application of SIK2 protein of grouper or the encoding gene of said SIK2 protein or a recombinant vector containing said encoding gene in the breeding of fish resistant to viral nerve necrosis.

5. The application according to any one of claims 1 to 4, characterized in that, The amino acid sequence of the SIK2 protein is shown in SEQ ID NO.1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2.

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