Application of Opnlrp12 gene in prevention and treatment of sparus punctatus virus and / or bacterial infection

Abstract

The invention provides an application of an Opnlrp12 gene in prevention and treatment of torus schlegeli virus and / or bacterial infection, and belongs to the technical field of gene engineering. Through targeted knockout of Opnlrp12, it is found that the expression level of a disease-resistant gene is also reduced, and therefore it is proved that Opnlrp12 can effectively inhibit infection caused by viruses and / or bacteria, and the survival rate of oplegnathus punctatus is increased. By means of the method, control over inflammatory diseases caused by iridovirus and / or vibrio harveyi can be achieved, and a target spot is provided for treatment of iridovirus infection of oplegnathus punctatus.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the application of the Opnlrp12 gene in the prevention and control of spotted sea bream virus and / or bacterial infection. Background Technology

[0002] Iridovirus is a cytoplasmic DNA virus. The first iridovirus was isolated by Xeros from the swamp mosquito in Cambridge, England in 1954. Because iridovirus particles are arranged in an unusually orderly, periodically spaced pattern within infected insect larvae or in purified, concentrated viral precipitates, forming overlapping lattice planes, they exhibit a blue or purple iridescence when illuminated by oblique light. Iridovirus primarily infects invertebrates and lower vertebrates, and its hosts include almost all major marine farmed fish and important freshwater farmed fish. This virus can cause mass mortality in farmed grouper, red sea bream, and other species, with mortality rates reaching 60% to 100%.

[0003] Nucleotide-binding oligomerization domain-like receptors (NLRs) are a class of proton pump receptors (PRRs) widely distributed in the cytoplasm, participating in the innate immune response by regulating inflammatory signaling pathways. NLRP12 is an important member of the NLR family, capable of targeting different inflammatory signaling pathways to exert regulatory effects. NLRP12 (NLR family PYRIN domain containing 12), also known as PYPAF7 (PYRIN-containing Apaf1-like protein 7), is a member of the NLRP subfamily, and its encoding gene is located on human chromosome 19. Like most NLRP receptors, NLRP12 consists of three major domains: the N-terminal PYRIN domain, the middle NACHT domain, and the C-terminal LRR domain. In vitro studies have shown that NLRP12 has a wide range of regulatory functions. On the one hand, NLRP12 can negatively regulate the classical / non-classical NF-κB signaling pathway and inhibit the production of pro-inflammatory cytokines and chemokines. NLRP12 effectively inhibits the activation of the classical NF-κB pathway mediated by Pam3Cys (TLR1 / 2 activator) or LPS by interacting with IRAK1 and inhibiting its phosphorylation. Simultaneously, NLRP12 can also interact with NIK, hydrolyzing NIK via the protease pathway, thereby significantly inhibiting the activation of the non-classical NF-κB pathway mediated by CD40. However, the function of NLRP12 in iridovirus infection has not been definitively reported. Summary of the Invention

[0004] The purpose of this invention is to provide the application of Opnlrp12 in the prevention and control of viral and / or bacterial infections in spotted sea bream, and to solve the problem of high mortality rate in spotted sea bream caused by iridovirus and Vibrio harveyi.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides the application of the Opnlrp12 gene in the prevention and control of spotted sea bream virus and / or bacterial infection. The coding region sequence of the Opnlrp12 gene is shown in SEQ ID NO. 1.

[0007] The coding region sequence of the Opnlrp12 gene:

[0008]

[0009] Preferably, the virus includes an iridovirus, and the bacteria includes Vibrio harveyi.

[0010] This invention provides the application of a functional product that regulates the expression level of the Opnlrp12 gene in the preparation of drugs for treating inflammatory diseases caused by iridovirus infection and / or Vibrio harveyi infection.

[0011] Preferably, the functional product has the function of downregulating the expression, transcription, or expression product of the Opnlrp12 gene.

[0012] Preferably, the functional product includes any one of the following: an antisense oligonucleotide, siRNA, ribozyme, shRNA, or gene editing vector capable of downregulating the expression of the Opnlrp12 gene.

[0013] This invention also provides the application of downregulating or knocking out the Opnlrp12 gene in reducing the expression of CD40, IL6, IFNα and IRF3 in the kidney cells of spotted sea bream.

[0014] The present invention also provides a method for knocking out the Opnlrp12 gene in the kidney cell line of spotted sea bream, wherein spotted sea bream kidney cells are transfected with siRNA, and the sequences of the sense strand and antisense strand of the siRNA are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively.

[0015] Preferably, the transfection system is: 1×CP buffer 60 μL, CP Regent 6 μL, siRNA 2.5 μL.

[0016] Preferably, the transfection time is 24 hours.

[0017] By adopting the above technical solution, the present invention has the following beneficial effects: By targeting and knocking out Opnlrp12, the expression level of disease-resistant genes was found to be reduced, thus proving that Opnlrp12 can effectively inhibit infections caused by viruses and / or bacteria, and improve the survival rate of spotted sea bream. The method of the invention can achieve the control of inflammatory diseases caused by iridovirus and / or Vibrio harveyi, providing a target for the treatment of iridovirus infection in spotted sea bream. Attached Figure Description

[0018] Figure 1 Phylogenetic tree analysis of nlrp12 of spotted rock seabream and nlrp12 of other species.

[0019] Figure 2The relative expression levels of nlrp12 in healthy tissues of spotted sea bream are shown. Different letters indicate significant differences in expression levels; where Gi represents gill tissue, Sk represents skin tissue, St represents stomach tissue, Li represents liver tissue, H represents heart tissue, K represents kidney tissue, Sp represents spleen tissue, In represents intestine tissue, HK represents head kidney tissue, and Br represents brain tissue.

[0020] Figure 3 The expression levels of nlrp12 in the liver (a), spleen (b), and kidney (c) at different time points after iridovirus infection are shown. *P<0.05, **P<0.01, ***P<0.001.

[0021] Figure 4 The expression levels of nlrp12 in the liver (a), spleen (b), and kidney (c) at different time points after Vibrio harveyi infection were shown. *P<0.05, **P<0.01, ***P<0.001.

[0022] Figure 5 The expression analysis of nlrp12 in OPK after 6 h of stimulation with different concentrations of poly I:C and LPS.

[0023] Figure 6 This is a graph showing the expression analysis of nlrp12 in OPK after treatment with three different siRNAs.

[0024] Figure 7 This is a graph showing the expression analysis of five immune-related genes in OPK after siRNA1 treatment. Detailed Implementation

[0025] 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.

[0026] The healthy spotted sea bream used in this experiment were all purchased from Shandong Laizhou Mingbo Aquatic Products Co., Ltd., and weighed about 120 g. They were temporarily kept in a water tank for 5 days before the experiment to ensure sufficient dissolved oxygen.

[0027] The spotted sea bream kidney cell line (Oplegnathus punctatus kidney, OPK) used in the immune stimulation experiment was obtained from the Aquatic Biotechnology and Genomics Laboratory of the Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences.

[0028] Example 1. Sequence analysis and phylogenetic tree of Opnlrp12 in *Scoidea spp.*

[0029] The cDNA of Opnlrp12 from *Syngonium spp.* was analyzed, and the amino acid sequence of the Opnlrp12 gene was predicted using DNAman software. The molecular weight and theoretical isoelectric point of the Opnlrp12 gene were predicted using SWISS-PROT (https: / / www.expasy.org / ). The results showed that the open reading frame of the cDNA of *Syngonium spp.*'s nlrp12 is 5295 bp, encoding 1764 amino acids, with a predicted protein molecular weight of 197.3 kDa and a theoretical isoelectric point of 6.58. SMART software predictions show that nlrp12 has one RING domain (amino acids 74-116), one FISNA domain (amino acids 147-219), and 29 LRR domains (located at amino acids 906-933, 935-962, 964-991, 994-1017, 1022-1049, 1051-1078, 1080-1107, 1109-1136, 1138-1165, 1168-1191, 1196-1220, 1225-1252, and 1...). Amino acids 254-1281, 1284-1307, 1312-1340, 1342-1369, 1371-1395, 1401-1424, 1429-1456, 1459-1482, 1487-1514, 1517-1540, 1545-1572, 1575-1598, 1603-1630, 1633-1656, 1661-1685, 1690-1717, and 1719-1746.

[0030] Homologous conserved functional domains were predicted using the SMART online website (https: / / smart.embl.de / smart / set_mode.cgi?NORMAL=1). The amino acid sequences of nlrp12 from different vertebrates were downloaded from NCBI and homology alignment was performed using BLAST. Multiple alignments of the nlrp12 amino acid sequences between species were performed using DNAman. BLAST alignment revealed that the amino acid sequence encoded by Opnlrp12 showed high similarity to the amino acid sequences encoded by nlrp12 from other bony fishes, with similarities of 75.20%, 77.93%, and 79.44% with Solea senegalensis, Channa argus, and Thunnus albacares, respectively. Similarity to mammals (Homo sapiens) and mice (Musmusculus) was lower, at 43.14% and 37.26%, respectively.

[0031] To investigate the role of Opnlrp12 in animal evolution, a phylogenetic tree of nlrp12 in mammals and bony fishes was constructed using the Neighbor-joining method in MAGE 7 software. The phylogenetic analysis showed that nlrp12 clustered into two branches: one with mammals (mice, cattle, humans, and chimpanzees) and another with other bony fishes. This indicates that Opnlrp12 is evolutionarily distantly related to mammals, but more closely related to bony fishes such as snakehead, albacore tuna, and Senegal sole.

[0032] Example 2. Expression of nlrp12 in various tissues of spotted sea bream

[0033] Four healthy spotted rock sea bream were randomly selected. After being anesthetized with MS-222, they were dissected and 10 types of tissues were collected, including liver, gills, heart, skin, spleen, intestine, brain, kidney, stomach, and head kidney. The tissues were quickly placed into cryovials containing RNA preservation solution and then flash-frozen in liquid nitrogen. After sampling, the samples were stored at -80°C for later use.

[0034] Using β-actin as an internal control, the expression level of Opnlrp12 in different tissues and organs of the experimental and control groups of spotted sea bream was detected by qRT-PCR. Each group had 4 replicates (N=4). The specific steps are as follows:

[0035] 1. Total RNA extraction from spotted sea bream

[0036] Total RNA was extracted using the Trizol method, and the steps are as follows:

[0037] (1) Add two sterile steel balls and 500 μL of Trizol to a 1.5 mL enzyme-free centrifuge tube. Cut about 30 mg of tissue with sterile scissors, blot the RNA preservation solution off the surface of the tissue with filter paper, and then put it into an enzyme-free centrifuge tube. Grind the tissue for 2 min at 60 Hz / min using a rapid grinder. Then add 500 μL of pre-cooled Trizol and shake well. Place the tube on ice for 10 min and manually shake it once every 3 min.

[0038] (2) Centrifuge at 12,000 rpm for 5 min in a 4℃ centrifuge, aspirate the supernatant and transfer it to a new 1.5 mL enzyme-free centrifuge tube; add 200 μL of pre-cooled chloroform, shake manually for 15 s, and place on ice for 10 min.

[0039] (3) Centrifuge at 12,000 rpm for 15 min in a centrifuge at 4℃. The liquid in the centrifuge tube is clearly divided into three layers: upper, middle and lower. Carefully aspirate the upper layer solution and transfer it to a new 1.5 mL enzyme-free centrifuge tube. Add 500 μL of pre-cooled isopropanol, shake well, and precipitate in a -20 ℃ refrigerator for 1 h.

[0040] (4) Centrifuge at 12,000 rpm for 10 min in a 4℃ centrifuge. The white RNA precipitate will be at the bottom of the centrifuge tube. Discard the supernatant and add 1 mL of pre-cooled 75% ethanol (prepared by mixing enzyme-free sterile water and anhydrous ethanol in a 1:3 ratio). Gently blow the precipitate to suspend it. Repeat 3 times.

[0041] (5) Centrifuge at 12,000 rpm for 5 min in a 4℃ centrifuge to remove as much residual ethanol as possible from the centrifuge tube. Then, leave the centrifuge tube cap open in a clean bench for 5 min to allow the residual ethanol to evaporate.

[0042] (6) Add 20 μL of pre-cooled enzyme-free water to the centrifuge tube to dissolve the RNA precipitate.

[0043] The relative ratios and concentrations of the extracted spotted sea bream RNA were determined using a DNA / Proteins Analyzer P100 instrument. The integrity of the extracted spotted sea bream RNA was detected by 1% agarose gel electrophoresis. High-quality RNA with good ratios and complete bands was then stored at -80°C.

[0044] 2. cDNA Synthesis

[0045] (1) Removal of genomic DNA

[0046] Prepare the following system in a 200 μL enzyme-free PCR tube: 1 μL gDNA Eraser, 1 μL 5× gDNA Eraser Buffer, 1 ng RNA, and RNase-free ddH2O to a final volume of 10 μL. Incubate at 42 °C for 2 min to remove genomic DNA.

[0047] (2) Reverse transcription reaction

[0048] Using extracted sea bream RNA as a template, the mRNA was reverse transcribed into cDNA using a first-strand synthesis kit and a reverse transcription kit. The reaction system is shown in Table 1.

[0049] Table 1 RT-PCR reaction system

[0050] reagents Dosage (μL) Spotted sea bream RNA extract 10 RNase Free ddH2O 4 5× PrimeScript Buffer 2 4 RT Primer Mix 1 PrimeScript RT Enzyme Mix I 1

[0051] RT-PCR reaction procedure: 37 ℃ for 15 min, 85 ℃ for 5 s.

[0052] The reverse transcription product was used with the β-actin F / R internal reference primer from the spotted sea bream to perform PCR to verify the reliability of the cDNA. The PCR reaction system is shown in Table 2.

[0053] β-actin F: 5'-GCTGTGCTGTCCCTGTA-3' (SEQ ID NO.3);

[0054] β-actin R: 5'-GAGTAGCCACGCTCTGTC-3' (SEQ ID NO. 4).

[0055] Table 2 PCR reaction system

[0056] reagents Dosage (μL) Spotted rock seabream cDNA 1 2× Taq Plus PCR Master Mix 10 <![CDATA[RNase Free ddH2O]]> 7 β-actin F 1 β-actin R 1

[0057] PCR program: 95 ℃ for 5 min, (95 ℃ for 30 s, 59 ℃ for 30 s, 72 ℃ for 30 s) × 34 cycles, 72 ℃ for 10 min.

[0058] The PCR products were detected by 1% agarose gel electrophoresis. The cDNA with a single and bright band was selected and stored at -20 ℃ for subsequent real-time quantitative PCR experiments.

[0059] 3. Real-time quantitative PCR

[0060] Primer design was performed using Primer 5.0 software.

[0061] nlrp12-RT-F: 5'-TCTCTGTGATCCACTGCTCA-3' (SEQ ID NO.5);

[0062] nlrp12-RT-R: 5'-AGTCTCTGTCGTCCCTCTTT-3' (SEQ ID NO. 6).

[0063] Using the cDNA of spotted sea bream obtained by reverse transcription as a template and β-actin as an internal reference gene, SYBR was used. ® The expression level of the nlrp12 gene in *Sinonovacula spp.* was determined using a Green ProTaq HS premixed qPCR kit (AG, China) strictly following the instructions on a 7500 FAST real-time PCR instrument (ABI, USA). The real-time PCR reaction system is shown in Table 3.

[0064] Table 3. Real-time PCR reaction system

[0065] reagents Dosage (μL) 2× SYBR Green Pro Taq HS Premix 10 cDNA 1 nlrp12-RT-F 0.8 nlrp12-RT-R 0.8 ROX Reference Dye 0.4 <![CDATA[RNase Free ddH2O]]> 7

[0066] The quantitative PCR reaction program was: 95 ℃ for 30 s, followed by (95 ℃ for 5 s, 60 ℃ for 34 s) × 40 cycles. Two [units / items] were used. –ΔΔCt The relative expression levels of the gene were calculated, and the relative expression levels of the nlrp12 gene in different tissues were plotted using Excel. SPSS software was used to test the significance of differences in gene expression levels. The results are as follows: Figure 2 As shown.

[0067] The results showed that Opnlrp12 was expressed in all 10 tissues of the spotted sea bream, with relatively high expression levels in the skin, stomach, liver, gills, and heart, slightly lower expression levels in the kidneys and spleen, and relatively low expression levels in the intestines, head kidneys, and brain.

[0068] Example 3. Expression analysis of Opnlrp12 gene in immune tissues after iridovirus infection.

[0069] Fifty healthy, well-grown spotted sea bream were randomly divided into two groups. Each fish in the experimental group was injected intraperitoneally with 100 μL of SKIV-SD iridovirus (10 μL / kg). 9 (Copy number), the control group received an equal volume of 1×PBS intraperitoneally. At five time points (0, 1, 4, 7, and 10 days) after injection, five spotted sea bream were randomly selected, anesthetized, and dissected. Three immune tissues (liver, spleen, and kidney) were collected. The tissues were quickly placed in cryovials containing RNA preservation solution, flash-frozen in liquid nitrogen, and stored at -80°C for later use.

[0070] The relative expression levels of Opnlrp12 in the liver, kidney, and spleen at different time points after intraperitoneal injection of iridovirus were detected using qRT-PCR. The PBS group had 4 replicates (N=4), and the iridovirus group had 5 replicates (N=5). The detection method was the same as in Example 2. The results are as follows: Figure 3 As shown in the figure. The results showed that Opnlrp12 responded rapidly to iridovirus, and the expression level of Opnlrp12 in the three immune tissues was significantly increased on day 0, which was 2.1 times, 2.1 times, and 2.7 times that of the control group, respectively. The expression level in the spleen was also significantly upregulated on days 1 and 4.

[0071] Example 4. Expression analysis of nlrp12 in immune tissues after Vibrio harveyi infection

[0072] Fifty healthy spotted sea bream in good condition were randomly divided into two groups. Each fish in the experimental group was injected intraperitoneally with 100 μL of Vibrio harveyi (1×10⁻⁶). 9 (cfu / mL), the control group was injected with an equal volume of 1×PBS, and then at 0 h, 12 h, 24 h, 48 h and 72 h after injection, 5 spotted sea bream were randomly selected, anesthetized and dissected, and 3 immune tissues of liver, spleen and kidney were taken. The tissues were quickly placed into cryovials containing RNA preservation solution, immediately immersed in liquid nitrogen for flash freezing, and then stored at -80℃ for later use.

[0073] Using β-actin as an internal control, the expression levels of Opnlrp12 in the liver, spleen, and kidney of the experimental and control groups of spotted sea bream were detected by qRT-PCR. Four replicates (N=4) were performed in the PBS group, and five replicates (N=5) were performed in the Vibrio harveyi group. The detection method was the same as in Example 2. The results are as follows: Figure 4 As shown.

[0074] The results showed that the expression levels of Opnlrp12 in the liver, spleen, and kidney were upregulated after stimulation with Vibrio harveyi, and the expression levels in all three tissues were significantly increased at 12 h and 24 h. In addition, the expression levels in the liver and spleen were also significantly upregulated at 48 h and 72 h, respectively.

[0075] Example 5. Analysis of the expression pattern of Opnlrp12 in renal cells after immune stimulation

[0076] To further investigate the role of Opnlrp12 in the antiviral and bacterial infection resistance of spotted sea bream, an in vitro stimulation experiment was designed using spotted sea bream kidney cell lines. Spotted sea bream brain cell lines were stimulated with different concentrations of poly I:C (0, 50, 100, 200 μg / mL) and LPS (0, 50, 100, 200 μg / mL). After 6 h, cells were collected, and cellular RNA was extracted to prepare a quantitative template. The expression level of Opnlrp12 after immunostimulation was detected by qRT-PCR. A control group of 0 μg / mL was used. The results are as follows: Figure 5 As shown.

[0077] The results showed that the expression level of Opnlrp12 first increased and then decreased with increasing poly I:C stimulation concentration; the expression level of Opnlrp12 in OPK was upregulated after stimulation with different concentrations of LPS. The expression level of Opnlrp12 was highest when the poly I:C and LPS stimulation concentrations were 50 μg / mL and 100 μg / mL, respectively, which were 1.6 times and 1.7 times that of the control group, respectively, indicating that immune stimulation can induce upregulation of Opnlrp12 expression.

[0078] Example 6. Knockdown of the Opnlrp12 gene in kidney cells affects the expression of four disease resistance genes.

[0079] Three siRNA sequences were designed based on the ORF region of the Opnlrp12 gene, namely siRNA1, siRNA2, and siRNA3. The siRNA sequences and primer sequences for disease resistance-related genes are shown in Table 4.

[0080] Table 4. Sequences and primers used in siRNA interference experiments

[0081]

[0082] The spotted sea bream kidney cell line used in the siRNA interference experiment was obtained from the Aquatic Biotechnology and Genomics Laboratory of the Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences. Monolayer cultured spotted sea bream kidney cells in good growth condition were trypsinized and seeded into 12-well plates. After 24 h, when the cell coverage reached approximately 60%, siRNA transfection was performed. The riboFECT™ CP transfection kit was used to transfect Opnlrp12 cells with siRNA at a final concentration of 50 nmol / L. The sense and antisense strands were mixed in equal proportions for each group. The transfection system per well consisted of: 60 μL of 1×CP buffer, 6 μL of CPRegent, and 2.5 μL of siRNA (siRNA1-3; NC; cy3). Each of the three siRNA sequences was repeated in 6 wells, NC in 4 wells, and cy3 in 2 wells for assessing fluorescence transfection efficiency. The cy3 wells were observed under a fluorescence microscope 24 h post-transfection to determine transfection efficiency. 72 h post-transfection, cells were collected using TRIzol, RNA was extracted, and reverse transcribed into cDNA. The knockdown effect of Opnlrp12 and the expression levels of immune-related genes were detected using quantitative real-time PCR. The experiment was repeated three times, and the quantitative real-time PCR method was consistent with that in Example 3.

[0083] Experiments showed that siRNA1 knockdown had the best effect, which was significantly different from the NC group. Figure 6 Treatment with siRNA1 resulted in downregulation of Opnlrp12, leading to a significant decrease in the expression levels of CD40, IL6, IFNα, and IRF3, while TBK1 levels showed no significant difference. This suggests that Opnlrp12 can regulate the expression of downstream disease-resistance genes and may play an important role in viral infection. Figure 7 ).

[0084] As can be seen from the above embodiments, the present invention provides the application of the Opnlrp12 gene in the prevention and control of spotted sea bream virus and / or bacterial infection. The Opnlrp12 gene can effectively identify virus / or bacterial resistance populations, providing a new target for the prevention and control of spotted sea bream iris virus and germplasm selection.

[0085] 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. Opnlrp12 Application of genes in the prevention and control of spotted sea bream virus and / or bacterial infections. Opnlrp12 The gene coding region sequence is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The virus includes iridovirus, and the bacteria include Vibrio harveyi.

3. Regulation Opnlrp12 Application of functional products with high gene expression levels in the preparation of drugs for inflammatory diseases caused by iridovirus infection and / or Vibrio harveyi infection.

4. The application according to claim 1, characterized in that, The functional products have a downward adjustment Opnlrp12 The function of gene expression, transcription, or its expression products.

5. The application according to claim 3 or 4, characterized in that, The functional product includes any one of the following: capable of lowering Opnlrp12 Gene expression can be achieved through antisense oligonucleotides, siRNA, ribozymes, shRNA, or gene editing vectors.

6. Downgrade or remove Opnlrp12 Application of gene therapy in reducing the expression of CD40, IL6, IFNα and IRF3 in kidney cells of spotted sea bream.

7. A knockout kidney cell line of spotted sea bream Opnlrp12 The gene-based method is characterized by, Kidney cells of spotted sea bream were transfected with siRNA, the sense and antisense strand sequences of which are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively.

8. The method according to claim 7, characterized in that, The transfection system consisted of: 1×CP buffer 60 μL, CP Regent 6 μL, and siRNA 2.5 μL.

9. The method according to claim 8, characterized in that, The transfection time was 24 hours.

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