Portunus trituberculatus miRNA novel294 and application thereof
By screening and chemically modifying miRNA novel294 from the tricuspid crab, an inhibitor with significant inhibition of the growth of Vibrio parahaemolytica was prepared, which solved the frequent disease problems caused by this bacterial infection, achieved significant inhibitory effect, and provided a new antibacterial strategy for the breeding industry.
Patent Information
- Application Number
- CN202410767271.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-06-14
- Publication Date
- 2025-05-09
AI Technical Summary
In the breeding of tricuspid supra crabs, diseases caused by Vibrio parahemolytic infection have occurred frequently, which seriously affects the development of the industry. There are few researches on related preparations directly applied to anti-bacterial agents in the existing technology.
By screening out miRNA novel294 from the scattered crab and chemically modifying it, an inhibitor with significant inhibition of Vibrio parahaemolytic growth was prepared.
Experimental verification shows that miRNA novel294 inhibitor can significantly inhibit the growth of Vibrio parahaemolyticus, with an inhibition rate of 26.8% in 9 hours, and has potential application value in disease prevention and treatment of serpent crabs, antibacterial drug development and feed additive production.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of molecular biology, and particularly relates to a swimming crab miRNA novel294 and an application thereof. Background Art
[0002] Swimming crab Portunus trituberculatus It belongs to the class Crustacea, order Decapoda, family Portunidae, genus Portunus, and is one of the leading species in marine crab farming in China. In 2022, the national swimming crab farming area was 23,000 hectares, with a production of 109,000 tons, which has important economic value.
[0003] At present, the breeding of swimming crabs mainly relies on large-scale outdoor pond breeding. Intensive breeding over generations has led to frequent diseases. Various diseases such as vibriosis and filamentous bacterial diseases have seriously affected the development of the swimming crab industry. Vibrio parahaemolyticus It is one of the main pathogens that causes frequent diseases of swimming crabs. After infection, it will lead to slow growth and mass death, causing significant economic losses. Vibrio parahaemolyticus belongs to the genus Vibrio and is a halophilic Gram-negative bacterium. Vibrio parahaemolyticus is an opportunistic pathogen. Current studies have shown that there are 8 virulence factors that are closely related to the pathogenicity of Vibrio parahaemolyticus, namely hemolysin, type III secretion system, type IV secretion system, adhesion factor, iron uptake system, lipopolysaccharide, protease and outer membrane protein. Vibrio parahaemolyticus is considered to be the pathogen that causes acute hepatopancreatic necrosis disease (AHPND) in aquatic animals.
[0004] miRNA is a highly conserved endogenous non-coding short-chain RNA with a length of about 22 nt. It plays a huge role in biological development, differentiation, apoptosis and stress resistance. Studies have shown that exogenous miRNA can inhibit the growth of viruses, bacteria and fungi, but its mechanism of action is not clear. At present, there are few studies on related preparations directly used against Vibrio parahaemolyticus. Summary of the invention
[0005] In view of the above technical problems, the present invention aims to provide a Swimming crab miRNA novel294 and its application. The present invention has experimentally verified that the miRNA novel294 has a significant inhibitory effect on the growth of Vibrio parahaemolyticus.
[0006] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions: The present invention provides a swimming crab miRNA novel294, whose nucleotide sequence is shown as SEQ ID NO.1.
[0007] The present invention also provides the use of the Swimming crab miRNA novel294 in preparing an inhibitor for inhibiting bacteria.
[0008] Further: the sequence shown in SEQ ID NO.1 is chemically modified to obtain G * G * U UCGAUUCCCGGCACGGG A * A * G , underlined bases indicate the use of 2'-Ome instead of 2'-OH; * indicates phosphorothioate.
[0009] Further: the bacterium is Vibrio parahaemolyticus.
[0010] Furthermore: the concentration of the inhibitor is 3~6 μM.
[0011] Furthermore: the inhibitor has the best inhibitory effect at 9 hours.
[0012] The advantages and technical effects of the present invention are as follows: the present invention provides miRNAnovel294 with a sequence as shown in SEQ ID NO.1, and chemically modifies the bases therein, and the miRNA novel294 provided by the present invention has a significant technical effect of inhibiting the growth of Vibrio parahaemolyticus through the inhibition experiment of Vibrio parahaemolyticus, and can be used to prepare an inhibitor of Vibrio parahaemolyticus, and the present invention has potential application value in disease prevention and control of swimming crabs, development of antibacterial drugs, production of feed additives, etc. The present invention also has important theoretical significance and application value for studying the immune defense mechanism of swimming crabs, disease prevention and control, and breeding of new disease-resistant varieties. BRIEF DESCRIPTION OF THE DRAWINGS
[0013] Figure 1 This is a graph showing the results of miRNA novel294 provided by the present invention inhibiting the growth of Vibrio parahaemolyticus (* P <0.05); novel294 indicates novel294 inhibitor and Vibrio parahaemolyticus functional incubation, and NC indicates NC and Vibrio parahaemolyticus co-incubation. Values are the average of three repeated experiments and are expressed as mean ± standard deviation. DETAILED DESCRIPTION
[0014] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods, but the scope of protection claimed by the present invention is not limited to the scope described in the examples.
[0015] Example 1 Obtaining the non-coding small RNA-miRNA novel294 from the swimming crab (Portunus trituberculatus): Based on miRNA sequencing analysis under Vibrio parahaemolyticus infection, the present invention obtained the differentially expressed miRNA novel294 using two softwares, miRanda and targetscan. The nucleic acid sequence of the miRNA is as follows: 5'-GGUUCGAUUCCCGGCACGGGAAG-3' (SEQ ID NO.1).
[0016] Example 2 Experiments using the miRNA novel294 as an inhibitor against Vibrio parahaemolyticus: 1. In order to increase the stability of miRNA, the novel294 sequence was synthesized in vitro by chemical synthesis. G * G * U UCGAUUCCCGGCACGGG A * A * G , and chemically modified (the underlined bases indicate the use of 2'-Ome to replace 2'-OH; * indicates phosphorothioate (bond replacing disulfide bond)); a control group (named NC) was also synthesized.
[0017] The miRNA novel294 inhibitor and control group NC were dissolved in DECP water to 100 μM, namely novel294 inhibitor solution and NC solution, respectively. The inhibitor and control group NC (U*U*GUACUACACAAAAGUAC*U*G) were synthesized by Sangon Biotech (Shanghai) Co., Ltd.
[0018] 2. Preparation of Vibrio parahaemolyticus suspension Weigh 2216E culture medium (Shenggong, Shanghai) and dissolve it with ultrapure water. Sterilize it at 121℃ in a high-temperature steam sterilizer for 15 min. After sterilization, place the liquid culture medium at 4℃ for use. Take out a small amount of Vibrio parahaemolyticus strain frozen in a -80℃ refrigerator and add it to 2216E culture medium; 28℃, 200rpm, 4-5h to obtain activated strains; take 100 μL of the activated strain and inoculate it in 100 mL culture medium, and culture it at 37℃, 200rpm overnight. Use an ELISA instrument to detect the concentration of bacteria and dilute the bacterial concentration to 10 5 CFU / mL.
[0019] 3. Co-incubation of miRNA and Vibrio parahaemolyticus The experimental group and the control group took 200 μL of 10 5CFU / mL bacterial solution was placed in a 96-well cell culture plate, with three replicates per group. 10 μL of the novel294 inhibitor solution from step 1 above was added to each well in the experimental group; the final concentration was 4.8 μM; 10 μL of the NC solution from step 1 above was added to each well in the control group, with a final concentration of 4.8 μM.
[0020] 4. Bacterial concentration detection The absorbance of each group in step 3 was recorded every 3 hours to detect the bacterial concentration. The results showed that after 3 hours of co-incubation, there was no significant difference in the number of bacteria between the experimental group and the control group; after 6 hours of co-incubation, the number of bacteria in the experimental group was significantly higher ( P <0.05) was lower than the control group, and the inhibition effect was the best at 9h, with an inhibition rate of 26.8% ( Figure 1 ). These results indicate that the miRNA novel294 inhibitor can significantly inhibit the growth of V. parahaemolyticus.
[0021] The above factual cases only represent the technical solutions of the present invention, rather than limiting the experiments. Although we have improved the experimental solutions, researchers in the same field can still make further improvements to the experimental solutions described above or make scientific equivalent replacements for the experimental links. These changes do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed to be protected by the present invention.
Claims
1. A miRNA novel294 from Portunus trituberculatus, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.
1.
2. Use of the Portunus trituberculatus miRNA novel294 according to claim 1 in the preparation of an inhibitor for inhibiting bacteria.
3. The use according to claim 2, characterized in that: The sequence shown in SEQ ID NO.1 was chemically modified to obtain G * G * U UCGAUUCCCGGCACGGG A * A * G , underlined bases indicate the use of 2'-Ome instead of 2'-OH; * indicates phosphorothioate.
4. The use according to claim 2, characterized in that: The bacterium is Vibrio parahaemolyticus.
5. The use according to claim 2, characterized in that: The concentration of the inhibitor was 3-6 μM.
6. The use according to claim 2, characterized in that: The inhibitor has the best inhibitory effect at 9 h.