Ruditapes philippinarum miR-263b inhibitor and application thereof

Abstract

The invention discloses a Ruditapes philippinarum miR-263b inhibitor and application thereof, and finds that Ruditapes philippinarum miR-263b can regulate and control innate immune response of a host by targeting RpSC2, negatively regulates activation of an immune-related signal channel in a vibrio infection process, and further, on the basis of a molecular regulation and control mechanism, the Ruditapes philippinarum miR-263b inhibitor can be used for inhibiting the infection of vibrio. The invention discloses a specific inhibitor which is completely complementary with a miR-263b mature body sequence. Experimental data show that after Ruditapes philippinarum is injected with the miR-263b inhibitor, the expression level of miR-263b in vivo is reduced, and the inhibition effect of miR-263b on RpSC2 is effectively relieved, so that the expression level of a target gene RpSC2 is up-regulated, an NF-kappa B immune signal channel is activated, the immune defense capability of the Ruditapes philippinarum on vibrio anguillarum is enhanced, the death rate after infection is remarkably reduced, and the Ruditapes philippinarum is prevented from being infected. The in-vivo bacterium loading amount is obviously reduced, and the pathological injury of hepatopancreas tissue is obviously relieved.

Description

A miR-263b inhibitor of the Manila clam and its application Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a miR-263b inhibitor of the Manila clam and its application. Background Technology

[0002] In aquaculture, the main cause of large-scale shellfish mortality is pathogenic microbial infection, with Vibrio infection being particularly serious. However, chemical control methods are prone to drug residues, environmental pollution, and antibiotic resistance in pathogens; vaccine control suffers from long development cycles and poor adaptability to variable pathogens.

[0003] MicroRNAs (miRNAs) are a class of endogenous non-coding small RNAs, approximately 18–25 nucleotides in length, widely distributed in eukaryotes. They can regulate gene expression at the post-transcriptional level by complementary binding to target gene mRNAs, playing a crucial role in organismal growth and development, immune responses, and pathogen defense. Studies have shown that miRNAs play an important role in regulating the innate immune response of aquatic animals, participating in the host's defense against pathogens by modulating the expression of immune-related genes. For example, CgIκB2 in oysters, as an important member of the NF-κB pathway, can mediate the target gene CgIκB2 by regulating the expression of miR-2d, thereby enhancing the phagocytosis of foreign bacteria. miR-145 regulates the inflammatory cytokine interleukin-17 (IL-17), enhancing the innate immune response of mussels (Mytilus coruscus). Furthermore, it has been found that the expression of apoptosis genes in oysters (Crassostrea gigas) after infection with Vibrio splendidus is closely related to miR-21-5p.

[0004] The core function of the peptidoglycan recognition protein (RpSC2) in the Manila clam (Ruditapes philippinarum) is to recognize peptidoglycan in the bacterial cell wall and participate in the innate immune response. Existing literature indicates that RpSC2 has significant inhibitory effects on various aquatic pathogens, including Vibrio anguillarum, Vibrio alginolyticus, and Vibrio parahaemolyticus; injection of recombinant RpSC2 protein can significantly improve the survival rate of clams infected with Vibrio anguillarum, increase hemocytocyte count, enhance the activity of antioxidant enzymes such as CAT and NOS, and reduce damage to the hepatopancreas and gill tissues. However, to date, there are no reports on the regulation of the host's innate immune response by Manila clam miRNAs through targeting RpSC2. Summary of the Invention

[0005] The present invention aims to solve the above-mentioned technical problems existing in the prior art by providing a miR-263b inhibitor of the Manila clam and its application.

[0006] The technical solution of the present invention is a miR-263b inhibitor of the Manila clam, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0007] The application of the above-mentioned miR-263b inhibitor of the Manila clam in the preparation of a drug for treating Vibrio anguillarum infection in Manila clam.

[0008] This invention discovers that miR-263b in Manila clams can regulate the host's innate immune response by targeting RpSC2, negatively regulating the activation of immune-related signaling pathways during Vibrio infection. Based on this molecular regulatory mechanism, a specific inhibitor completely complementary to the mature miR-263b sequence was invented. Experimental data show that after injection of the miR-263b inhibitor into Manila clams, the expression level of miR-263b in vivo was downregulated, effectively relieving the inhibitory effect of miR-263b on RpSC2, thereby upregulating the expression level of the target gene RpSC2, activating the NF-κB immune signaling pathway, enhancing the immune defense ability of Manila clams against Vibrio anguillarum, significantly reducing the mortality rate after infection, significantly decreasing the bacterial load in vivo, and significantly alleviating pathological damage to hepatopancreatic tissue. This provides a safe, efficient, and environmentally friendly technical solution for the prevention and control of diseases in Manila clams. Attached Figure Description

[0009] Figure 1 is an SDS-PAGE electrophoresis diagram of the recombinant protein of the target gene RpSC2 in an embodiment of the present invention.

[0010] Figure 2 shows the binding site of miR-263b of the Manila clam to the target gene RpSC2 and the experimental results of the dual-luciferase reporter gene assay in an embodiment of the present invention.

[0011] Figure 3 is a comparison of mortality rates of Manila clams infected with Vibrio anguillarum after being injected with RpSC2 recombinant protein according to an embodiment of the present invention.

[0012] Figure 4 shows the change in Vibrio anguillarum bacterial load in Manila clams after injection of RpSC2 recombinant protein in an embodiment of the present invention.

[0013] Figure 5 is a pathological section of the hepatopancreas tissue of the Manila clam after injection of RpSC2 recombinant protein in an embodiment of the present invention.

[0014] Figure 6 shows the changes in the expression levels of miR-263b and RpSC2 in Manila clams after injection of miR-263b-antagomir in an embodiment of the present invention.

[0015] Figure 7 is a comparison of the mortality rates of Manila clams under Vibrio anguillarum and LPS stress after injection of miR-263-antagomirb in an embodiment of the present invention.

[0016] Figure 8 shows the changes in the expression levels of miR-263b and RpSC2 in Manila clams after injection of miR-263b-agomir in an embodiment of the present invention.

[0017] Figure 9 is a comparison of the mortality rates of Manila clams under Vibrio anguillarum and LPS stress after injection of miR-263b-agomir according to the present invention. Detailed Implementation

[0018] The miR-263b inhibitor of the Manila clam of the present invention has a nucleotide sequence as shown in SEQ ID NO: 1, which is an antisense oligonucleotide that is completely complementary to the mature nucleotide sequence of the Manila clam miR-263b shown in SEQ ID NO: 2. It is prepared according to the following steps: Step 1. Using automated solid-phase oligonucleotide synthesis technology, with a solid-phase support as the substrate, antisense nucleoside monomers are sequentially linked in a stepwise extension manner according to the nucleotide sequence shown in SEQ ID NO: 1 to construct a complete antisense oligonucleotide chain; Step 2. The synthesized oligonucleotide is cleaved from the solid-phase support, and all protecting groups are removed to obtain a crude miR-263b inhibitor in free form; Step 3. The crude miR-263b inhibitor is purified by ion-exchange high-performance liquid chromatography, separating the full-length oligonucleotide and truncated fragments according to retention time, collecting and concentrating the components corresponding to the target peak to obtain a high-purity miR-263b inhibitor.

[0019] Experiment: 1. Preparation of experimental materials 1.1 Manila clam RpSC2, whose nucleotide sequence is shown in SEQ ID NO: 3. The recombinant RpSC2 protein was prepared as follows: RNA was extracted from Manila clam and reverse transcribed to synthesize cDNA; using the obtained cDNA as a template, a PCR reaction was performed, and the nucleotide sequences of the upstream and downstream primers for the PCR reaction were shown in SEQ ID NO: 4-5; the PCR reaction conditions were: denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30 s, annealing at 58 ℃ for 30 s, extension at 72 ℃ for 2 min, for 35 cycles; extension at 72 ℃ for 5 min; the amplified fragment with a length of approximately 1600 bp was purified and recovered, and ligated to a cloning vector using a ligase. After transformation into TOP10 competent cells, positive clones were screened, and plasmids were extracted; the plasmid was digested with NcoI / XhoI, and the target fragment generated by the digestion was purified and recovered; the purified target fragment was ligated with the NcoI / XhoI-digested expression vector pET-28a (+) in a medium containing 30 µg / ml kanamycin and 34 µg / ml... Cells were cultured in chloramphenicol medium. When the OD value reached 0.6, 0.5 mM IPTG was added, and the cells were cultured overnight at 20°C. The cells were then collected by centrifugation. The cells were dissolved in buffer C, sonicated, and the supernatant crude protein was collected by centrifugation. A 5 ml Ni-NTA column was used, and the column was equilibrated with 5 column volumes of binding buffer at a flow rate of 5 ml / min. The crude protein was incubated with the equilibrated column packing for 1 h before loading onto the column. The column was then equilibrated with binding buffer. After washing the column with washing buffer, the column was eluted with 20 mM Imidazole, and the eluent was collected. The eluent was dialyzed into a protein preservation buffer consisting of 50 mM Tris, 300 mM NaCl, 0.1% SKL, 2 mM DTT, and pH 8.0. After dialysis, the buffer was concentrated with PEG20000, filtered through a 0.45 μm filter, and stored. The amino acid sequence of the prepared recombinant RpSC2 protein is shown in SEQ ID NO: 6. SDS-PAGE was used to identify the protein purity, and the results are shown in Figure 1. Figure 1 shows that the target protein band is clear, and the molecular weight is consistent with the theoretical value.

[0020] 1.2 To improve the stability of miR-263b inhibitors in shellfish, 2′-O-methyl groups, locked nucleic acids, or phosphorus-sulfur bonds can be introduced onto their ribonucleotides for modification. In this embodiment of the invention, a 2′-O-methyl group is introduced onto the miR-263b inhibitor ribonucleotide to prepare a miR-263b antagonist (miR-263b-antagomir). Similarly, the unmodified sequence of the control sequence (NC-antagomir) is shown in SEQ ID NO: 7; the unmodified forward and reverse strand sequences of the miR-263b agonist (miR-263b-agomir) are shown in SEQ ID NO: 8-9, respectively; and the unmodified forward and reverse strand sequences of the control sequence (NC-antagomir) are shown in SEQ ID NO: 10-11, respectively. The specific application method is to prepare the sequence into a transfection solution. The transfection solution is prepared by mixing 20 μM antagonist or agonist working solution, Lipo6000 transfection reagent and PBS at a volume ratio of 1:1:8. The single injection volume is 100 μL / animal.

[0021] 1.3 Vibrio anguillarum (CICC 10475) was cultured in a shaker at 37°C for 12 h, centrifuged at 8000 rpm for 5 min, and then incubated with PBS at 5 × 10⁻⁶ ppm. 7 The concentration was diluted to CFU / mL, and the Vibrio anguillarum strain used was purchased from the China General Microbiological Culture Collection Center.

[0022] 2. Experimental Methods and Results 2.1 Target Gene Prediction and Validation of miR-263b: Potential target genes of miR-263b were predicted using software such as TargetScan and miRanda, and complementary binding sites with the RpSC2 gene were found. The targeted regulatory effect of miR-263b on RpSC2 was verified using a dual-luciferase reporter gene assay: A dual-luciferase reporter vector was constructed, and the 3'UTR fragments of wild-type and mutant RpSC2 were cloned into the reporter vector. The vector was then co-transfected with miR-263b-antagomir, NC-antagomir, miR-263b-agomir, and NC-agomir, respectively, into HEK-293T cells, and luciferase activity was detected. The results are shown in Figure 2.

[0023] In Figure 2A, the red areas represent the specific binding sites of miR-263b and RpSC2 at their 3'UTR.

[0024] Figure 2B shows that the WT group of miR-263b-agomir (red bar) showed a significant decrease in luciferase activity (approximately 0.5 vs 1.0) compared to NC-agomir (black bar), demonstrating that miR-263b can inhibit target gene expression through its binding site.

[0025] Compared with NC-antagomir (gray column), miR-263b-antagomir (blue column) showed a significant increase in luciferase activity (approximately 1.5 vs 1.0), further validating the negative regulatory effect of miR-263b on the target gene.

[0026] Figure 2B shows that the MT group did not show significant changes in luciferase activity regardless of whether it was transfected with an agonist or an antagonist, indicating that miR-263b could not play a regulatory role after the binding site mutation, which directly proves the specificity of the binding.

[0027] 2.2 Immunoprotective effect verification experiment of RpSC2 recombinant protein: Healthy Manila clams of uniform size were randomly divided into 4 groups of 100 clams each: PP group: injected with 100 μL PBS + 100 μL PBS; VP group: injected with 100 μL 5×10 7 CFU / mL Vibrio anguillarum bacterial culture + 100μL PBS; SP group: 100μL 50μg / mL RpSC2 recombinant protein + 100μL PBS; SV group: 100μL 50μg / mL RpSC2 recombinant protein + 100μL 5×10 7 CFU / mL Vibrio anguillarum bacterial suspension.

[0028] Experimental procedure: The mortality of clams in each group was recorded every 24 hours after injection. Hepatopancreatic tissues were collected at 0, 12, 24, 48, 72, 96 and 120 hours after injection to detect the amount of Vibrio anguillarum in the tissues and to prepare pathological sections to observe tissue damage.

[0029] Experimental results: Compared with the VP group, the mortality rate of Manila clams in the SV group was significantly reduced (Figure 3), the amount of Vibrio anguillarum in the hepatopancreas tissue was significantly reduced (Figure 4), and the histopathological damage was significantly reduced (Figure 5), indicating that the recombinant protein RpSC2 has a significant immunoprotective effect.

[0030] 2.3 Activity verification and interference efficiency detection of miR-263b inhibitor: Healthy Manila clams were randomly divided into an experimental group and a control group, with 40 clams in each group. The experimental group was injected with 100 μL of transfection solution containing miR-263b-antagomir, while the control group was injected with an equal amount of transfection solution containing the negative control sequence NC-antagomir. Hepatocellular and pancreatic tissues were collected at 0, 12, 24, 48, and 72 h after injection, and the expression levels of miR-263b and RpSC2 were detected by qPCR.

[0031] The results are shown in Figure 6: Compared with the control group, the expression level of miR-263b in the experimental group was significantly downregulated and the expression level of RpSC2 was significantly upregulated, indicating that miR-263b inhibitors can effectively interfere with the expression of miR-263b and relieve its inhibitory effect on the target gene RpSC2.

[0032] 2.4 Validation Experiment of the Anti-Vibrio Infection Efficacy of miR-263b Inhibitor: Healthy Manila clams of uniform size were randomly divided into 4 groups of 100 clams each: PBS group: injected with 100 μL transfection solution (without inhibitor) + 100 μL PBS; Inhibitor group: injected with 100 μL transfection solution containing miR-263b-antagomir + 100 μL PBS; Vibrio group: injected with 100 μL transfection solution (without inhibitor) + 100 μL 5×10 7 CFU / mL Vibrio anguillarum culture; Inhibitor + Vibrio group: Injection of 100 μL transfection solution containing miR-263b-antagomir + 100 μL 5×10 7 CFU / mL Vibrio anguillarum bacterial suspension.

[0033] Experimental procedure: 24 hours after injection of the transfection solution, Vibrio anguillarum infection was performed, and the mortality of clams in each group was recorded every 24 hours for a total of 168 hours. At the same time, an LPS stress group was set up to verify the protective effect of the inhibitor against lipopolysaccharide stimulation of Gram-negative bacteria.

[0034] The results are shown in Figure 7: Compared with the Vibrio group, the mortality rate of Manila clams in the inhibitor + Vibrio group was significantly reduced (Figure 7 left); under LPS stress, the mortality rate of the inhibitor treatment group was also significantly lower than that of the control group (Figure 7 right), indicating that miR-263b inhibitor can effectively enhance the resistance of Manila clams to Vibrio anguillarum infection.

[0035] 2.5 Validation of miR-263b overexpression: Healthy Manila clams were randomly divided into an experimental group and a control group, with 40 clams in each group. The experimental group was injected with 100 μL of transfection solution containing miR-263b-agomir, while the control group was injected with an equal volume of transfection solution containing the negative control NC-agomir. Hepatopancreatic tissues were collected at different time points after injection, and the expression levels of miR-263b and RpSC2 were detected by qPCR.

[0036] Anti-Vibrio infection effect: 24 hours after miR-263b overexpression, clams in each group were infected with Vibrio anguillarum and subjected to LPS stress, and the mortality rate was recorded.

[0037] The results showed that compared with the control group, the expression level of miR-263b in the experimental group was significantly upregulated (Figure 8 left) and the expression level of RpSC2 was significantly downregulated (Figure 8 right). Under Vibrio anguillarum infection (left) and LPS (right) stress, the mortality rate of the experimental group was significantly increased (Figure 9), which further verified that miR-263b negatively regulates the anti-Vibrio immune response of Manila clams by inhibiting RpSC2 expression.

Claims

1. A miR-263b inhibitor for Manila clams, characterized in that: Its nucleotide sequence is shown in SEQ ID NO:

1.

2. The use of the miR-263b inhibitor of the Manila clam as described in claim 1 in the preparation of a drug for treating Vibrio anguillarum infection in Manila clams.

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