Crassostrea gigas non-coding small RNA (miR-96a-5p) and application thereof in bacterial infection resistance
By designing miR-96a-5p inhibitors, Viagra splendid proliferation in the body of long oysters, the problem of prevention and control of bacterial infection by long oysters was solved, and the effect of effectively resisting bacterial infection was achieved.
Patent Information
- Application Number
- CN202411017596.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-07-29
- Publication Date
- 2024-12-17
AI Technical Summary
The prior art lacks effective prevention and control methods to deal with the deaths caused by bacterial pathogen infections of oysters, especially Vitiligo, caused by infection.
By designing and applying inhibitors of long oyster non-encoding small RNA miR-96a-5p, it inhibits its significantly reduced expression level after Viagra infection, thereby inhibiting the proliferation of Viagra in long oysters, and preparing anti-Vagra anti-Vagra anti-Vagra anti-Vagra anti-Vagra anti-Vagra anti-Vagra anti-Vagra anti-Vagra anti-Vagra anti-Vagra anti-Vagra anti-Vagra anti-Vagra anti-Vagra anti-Vagra Infection preparation.
The miR-96a-5p inhibitor significantly reduced the number of Vibrio splendid in the hemolymph of the long oyster, effectively resisted bacterial infection, and proved its application potential in anti-bacterial infection.
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Figure CN119144602A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of molecular biology, in particular to a non-coding small RNA (miR-96a-5p) of an oyster and its application in resisting bacterial infection. Background Art
[0002] The giant oyster (Crassostrea gigas) is one of the most widely farmed marine mollusks in the world. It not only has high economic value, but also has important ecological value. However, in recent years, large-scale mortality events have often occurred in farmed giant oysters, among which bacterial pathogen infection is one of the important causes of large-scale mortality. Vibrio plendidus is one of the main pathogens of giant oysters. After massive proliferation and infection, it can cause inflammatory reactions, tissue necrosis and even death in giant oysters. At present, there is still a lack of effective prevention and control methods for Vibrio plendidus infection.
[0003] miRNA is a small non-coding RNA that regulates gene expression by complementary binding to target gene mRNA. In most cases, miRNA induces degradation of target gene mRNA or prevents protein synthesis at the post-translational level. Studies have shown that miRNA plays an important role in regulating immune responses and anti-bacterial infection. In vertebrates, miR-146, miR-155, miR-125, let-7 and miR-21 regulate inflammatory responses during bacterial infection in humans and participate in the immune response process. In invertebrates, for example, cgi-miR-2d in long oysters regulates the phagocytosis of blood lymphocytes by targeting CgIκB2 in the nuclear factor (NF-κB) pathway, indicating that miRNA is involved in regulating the anti-bacterial infection and immune response process of invertebrates. At present, more and more miRNAs functions have been revealed, and their inhibitors or mimics also play an important role in regulating biological physiological metabolism and disease resistance. For example, an AMO-miRNA used for Japanese shrimp to resist WSSV infection can significantly inhibit the proliferation of WSSV in Japanese shrimp, effectively solving the phenomenon of death caused by WSSV infection in farmed shrimp. miR-96a-5p is a relatively conserved miRNA found in long oyster tissues and exosomes. Studies have shown that human miR-96-5p can regulate LPS-mediated inflammatory responses and cell apoptosis and improve the viability of LPS-treated HSAEC cells. In addition, miR-96-5p inhibitors can block human CRC cell migration and participate in cancer regulation. However, there is no research on the application of long oyster miR-96-5p inhibitors. Summary of the invention
[0004] The invention aims to provide a long oyster non-coding small RNA (miR-96a-5p) and its application in resisting bacterial infection.
[0005] To achieve the above purpose, the present invention adopts the following technical solution:
[0006] The invention discloses a non-coding small RNA (miR-96a-5p) of Crassostrea gigas. The non-coding small RNA (miR-96a-5p) of Crassostrea gigas is shown by the bases in SEQID No.1.
[0007] SEQ ID No.1
[0008] 5'-GUGACUAUUCCCGCCAGUGCCAAG-3'.
[0009] An application of the long oyster non-coding small RNA (miR-96a-5p), and an application of the long oyster non-coding small RNA (miR-96a-5p) in resisting bacterial infection.
[0010] The invention discloses an application of the long oyster non-coding small RNA (miR-96a-5p) in preparing an anti-bacterial infection preparation.
[0011] The invention discloses an application of the non-coding small RNA (miR-96a-5p) of Crassostrea gigas in the preparation of an anti-Vibrio splendens infection preparation.
[0012] The dosage of the anti-Vibrio splendens infection preparation is 100 μL / animal, and the concentration of miR-96a-5p in the preparation is 20 μM.
[0013] A preparation for Crassostrea gigas against Vibrio splendidus infection, comprising Crassostrea gigas non-coding small RNA (miR-96a-5p) shown in SEQ ID No. 1.
[0014] The present invention studies the expression of miRNAs in long oysters during Vibrio splendens infection, and finds that the expression level of miR-96a-5p in the blood lymphocytes of long oysters is significantly decreased after Vibrio splendens infection, indicating that miR-96a-5p may be involved in the long oysters' resistance to Vibrio splendens infection. The present invention designs an antisense miRNA inhibitor of miR-96a-5p to inhibit the expression level of miR-96a-5p. After the long oysters were injected with the miR-96a-5p inhibitor and stimulated with Vibrio splendens, the number of Vibrio splendens in the long oysters' blood lymph was significantly decreased compared with the negative control NC group injected with the miR-96a-5p inhibitor. The results show that the miR-96a-5p inhibitor can resist bacterial infection by inhibiting the proliferation of Vibrio splendens in the long oysters. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1The result graph of the miR-96a-5p inhibitor provided in the embodiment of the present invention inhibiting the proliferation of Vibrio splendens (*p<0.05). The blank control group was not treated, and the long oyster was stimulated with Vibrio splendens in the presence of the miR-96a-5p inhibitor negative control NC or in the presence of the miR-96a-5p inhibitor, and the number of Vibrio splendens in the hemolymph was detected at 12 hours. The values are the average of three experiments, expressed as mean ± standard deviation. DETAILED DESCRIPTION
[0016] The specific implementation modes of the present invention are further described below in conjunction with examples. It should be noted that the specific implementation modes described here are only for illustrating and explaining the present invention, and are not limited to the present invention.
[0017] The invention uses the miR-96a-5p inhibitor to effectively inhibit the proliferation of Vibrio splendens in the long oyster, and effectively resists the infection of Vibrio splendens to the long oyster during the breeding process.
[0018] Example 1
[0019] Obtaining the non-coding small RNA—miR-96a-5p from Crassostrea gigas:
[0020] miR-96a-5p is a non-coding small RNA expressed in the long oyster. Its sequence was obtained by nucleic acid sequencing as follows:
[0021] SEQ ID NO:1:5'-CUUGGCACUGCGGAAUAGUCAC-3'
[0022] Example 2
[0023] Experiment on miR-96a-5p inhibitor against Vibrio splendens infection:
[0024] Step 1) Preparation of miR-96a-5p inhibitor and miR-96a-5p inhibitor NC (negative control):
[0025] According to the sequence obtained in the above example, a miR-96a-5p inhibitor was designed, and its control sequence (named NC) was designed at the same time; the miR-96a-5p inhibitor and NC were dissolved in DEPC water (purchased from Jima Gene Co., Ltd.) to 10 μM respectively and set aside.
[0026] Among them, the miR-96a-5p inhibitor sequence is: 5'-GUGACUAUUCCGCCAGUGCCAAG-3'
[0027] The NC sequence is: 5′-CAGUACUUUUUGUGUAGUACAA-3.
[0028] Step 2) Preparation of Vibrio splendens injection
[0029] The Vibrio splendens used in this study was a pure strain stored at -80°C in the laboratory (containing a final concentration of 50% glycerol). The strain was placed on ice to thaw naturally, and then 2216E sterile seawater liquid culture medium was added and cultured in a constant temperature shaking incubator at 28°C for 24 hours to obtain bacteria in the logarithmic growth phase. The bacteria were collected by centrifugation at 3000×g for 10 minutes, and the residual culture medium was washed away with sterile seawater, and the concentration was adjusted to 1×10 7 CFU / mL is reserved.
[0030] Step 3) Injection into the oyster
[0031] Twenty-seven long oysters (each weighing about 90-110 g) were randomly divided into three groups, named A, B and C, with 9 in each group. Group A was the blank control group and was not treated. The long oysters in groups B and C were injected with 100 μL of miR-96a-5p inhibitor NC (negative control, 20 μM) and miR-96a-5p inhibitor (20 μM), respectively. 12 hours later, the long oysters in groups B and C were injected with 100 μL of Vibrio splendens (1×10 7 CFU / mL).
[0032] Step 4) Detection of Vibrio splendens
[0033] 12 h after the injection of Vibrio splendens in step 3, equal amounts of hemolymph from oysters from groups A, B, and C were collected and then 4 The samples were diluted by 100 times and plated on TCBS agar medium. After culturing in a 16°C incubator for 24 hours, the colonies were counted. The results showed that the number of Vibrio splendens in the hemolymph of oysters in group C injected with miR-96a-5p inhibitor was significantly reduced ( Figure 1 ), which was 0.59 times that of group B (p<0.001).
[0034] The above results indicate that miR-96a-5p inhibitors can resist bacterial infection by inhibiting the proliferation of Vibrio splendens in oysters.
Claims
1. A long oyster non-coding small RNA (miR-96a-5p), characterized by: The non-coding small RNA (miR-96a-5p) of Crassostrea gigas is shown by the bases in SEQ ID No.
1.
2. An application of the long oyster non-coding small RNA (miR-96a-5p) according to claim 1, characterized in that: The application of the long oyster non-coding small RNA (miR-96a-5p) in resisting bacterial infection.
3. The use according to claim 2, characterized in that: The invention discloses an application of the long oyster non-coding small RNA (miR-96a-5p) in preparing an anti-bacterial infection preparation.
4. The use according to claim 3, characterized in that: The invention discloses an application of the non-coding small RNA (miR-96a-5p) of Crassostrea gigas in the preparation of an anti-Vibrio splendens infection preparation.
5. The use according to claim 4, characterized in that: The dosage of the anti-Vibrio splendens infection preparation is 100 μL / animal, and the concentration of miR-96a-5p in the preparation is 20 μM.
6. A preparation for oysters against Vibrio splendidus infection, characterized in that: The preparation contains the long oyster non-coding small RNA (miR-96a-5p) shown in SEQ ID No. 1 described in claim 1.