WO2022241922A1

WO2022241922A1 - Mir-210 mimic, and preparation method therefor and use thereof as antibiotic substitute for culture

Info

Publication number: WO2022241922A1
Application number: PCT/CN2021/104504
Authority: WO
Inventors: 湛垚垚; 常亚青; 刘丽; 赵谭军; 宋坚
Original assignee: 大连海洋大学
Priority date: 2021-05-20
Filing date: 2021-07-05
Publication date: 2022-11-24
Also published as: CN113201544A

Abstract

A miR-210 mimic, wherein the RNA sequence of the miR-210 mimic is 5'-U SU SGUGCGUGCGACAGCGAC SU SG SA S-Chol-3'. The preparation method is carried out in sequence according to the following steps: using an RNA sequence of adult miR-210 to synthesize an oligonucleotide single strand, wherein the RNA sequence of the adult miR-210 is as shown in SEQ ID NO: 1; performing a cholesterol modification on the 3' end of the oligonucleotide single strand; performing two thio-backbone modifications on the 5' end of the oligonucleotide single strand, performing four thio-backbone modifications on the 3' end of the oligonucleotide single strand, and performing a methoxyl modification on the whole oligonucleotide single strand; and purifying the product to obtain the miR-210 mimic having the RNA sequence as shown in SEQ ID NO: 1. The miR-210 mimic can be used as a drug for inhibiting pathogens of skin ulceration syndrome in a Apostichopus japonicus culture or as a drug for inhibiting pathogens of red spotting disease in a sea urchin culture.

Description

miR-210 mimic, preparation method and application as a substitute for antibiotics for farming

technical field

The invention relates to the field of aquatic animal disease prevention and control technology and the development of environment-friendly antibiotic drug substitutes, in particular to a miR-210 simulant, a preparation method and its application as an antibiotic substitute for breeding.

Background technique

Aquaculture diseases caused by pathogenic bacteria have always been the core problem restricting the development of my country's aquaculture industry. At present, the main countermeasure is to use various antibiotics. Due to the non-standard use of antibiotics, strong drug-resistant and multi-drug-resistant pathogens in aquaculture have resulted in the use of antibiotics in aquaculture with higher and higher dosages and more and more species. Studies have shown that only about 20% of the antibiotics put into the water body in the aquaculture industry are used by organisms, and the residual antibiotics exist in the water body for a long time, which has a great impact on the quality of the water body, the ecological diversity of the water area, and the distribution and abundance of biological populations, etc. All will have profound and complex impacts. In addition, the antibiotics currently used in aquaculture are common drugs for humans and animals, and their antibiotic residues will also pose a potential threat to human health. Therefore, the development of environmentally friendly and efficient alternatives to antibiotics is one of the urgent needs to promote green and healthy aquatic farming.

microRNAs (miRNAs) are a class of endogenous small molecule non-coding RNAs (non-coding RNAs) consisting of 21-25 nucleotides that commonly exist in animals and plants. 3'Untranslated Regions (3'UTR) combined with the seed region (seed region) to degrade miRNA or inhibit the translation of related proteins, thereby regulating the expression of target genes at the post-transcriptional level. miRNAs mimics are artificially synthesized small molecule compounds that regulate the biological functions of target genes by simulating endogenous miRNAs in the body, thereby playing an important regulatory role in various life activities in the body and affecting the environment and human health. No effect. There have been no reports on the use of miRNAs mimics as antibiotic substitutes in the research of aquatic animal disease control, and thus applied to the development of green, healthy, pollution-free economical aquatic animal antibacterial drugs.

Contents of the invention

The present invention aims to solve the above-mentioned technical problems existing in the prior art, and provides a miR-210 simulant, a preparation method and an application as an antibiotic substitute for breeding.

The technical solution of the present invention is: a miR-210 mimic, characterized in that: the RNA sequence of the miR-210 mimic is 5'-U _S U _S GUGCGUGCGACAGCGAC _S U _S G _S A _S -Chol-3 '.

A method for preparing the above-mentioned miR-210 simulants, followed by the following steps:

Step 1: using the RNA sequence of sea cucumber or sea urchin adult miR-210 to synthesize oligonucleotide single strands, the RNA sequence of the adult miR-210 is shown in SEQ ID NO.1;

Step 2: Carrying out cholesterol modification on the 3' end of the oligonucleotide single strand;

Step 3: Carry out two thio-skeleton modifications to the 5' end of the oligonucleotide single-strand, perform four thio-skeleton modifications to the 3'-end of the oligonucleotide single-strand, and modify the oligonucleotide single-stranded whole chain carry out methoxy modification;

Step 4: Purify the product to obtain a miR-210 mimic whose RNA sequence is 5'-U _S U _S GUGCGUGCGACAGCGAC _S U _S G _S A _S -Chol-3'.

The application of the above-mentioned miR-210 simulant as a substitute for antibiotics for breeding is the application as a drug for inhibiting the pathogenic bacteria of rot skin syndrome for culturing sea cucumbers or as a drug for inhibiting the pathogenic bacteria of erythema for culturing sea urchins.

The invention adopts the miR-210 sequence of adult sea urchins or sea urchins to prepare miR-210 mimics, which is simple in vitro synthesis and convenient to use. In particular, the miR-210 used is different from the miR-210 sequence in other species including humans, will not interfere with the normal gene expression of other organisms, and will not affect ecological diversity and human health. By transfecting miR-210 mimics into the coelom fluid of sea cucumbers and sea urchins, the phagocytic activity of sea cucumbers and sea urchins can be significantly improved, so miR-210 mimics can replace antibiotics to play the role of antibacterial drugs, avoiding the use of antibiotics various ills.

Description of drawings

Fig. 1 is a schematic diagram of the sequence structure of the miR-210 mimetic of the embodiment of the present invention.

Figure 2 is a schematic diagram of the morphology of phagocytic cells and the phagocytosis process of Apostichopus japonicus.

Figure 3 is a schematic diagram of the experimental results of the effect of miR-210 mimics on the phagocytosis of Apostichopus phagocytic cells after infection with the pathogenic bacteria Vibrio splendidus of putrefaction syndrome.

Figure 4 is a schematic diagram of the experimental results of the effect of transfection of miR-210 mimics on the phagocytosis rate of sea cucumber phagocytic cells after being infected with putrefaction syndrome pathogenic bacteria Vibrio splendidus.

Fig. 5 is a schematic diagram of the phagocyte morphology and phagocytosis process of sea urchin mesenterosus.

Figure 6 is a schematic diagram of the experimental results of the effect of transfection of miR-210 mimics on the phagocytic ability of phagocytic cells of sea urchins mesenterus after infection with Vibrio erythematosus.

Figure 7 is a schematic diagram of the experimental results of the effect of transfection of miR-210 mimics on the phagocytosis rate of phagocytic cells of sea urchins mesenterus after infection with Vibrio erythematosus.

Detailed ways

The preparation method of the miR-210 mimic of the present invention is carried out in accordance with the following steps:

Step 1: using the RNA sequence of sea cucumber or sea urchin adult miR-210 to synthesize a single-stranded oligonucleotide, the RNA sequence of the adult miR-210 is shown in SEQ ID NO.1;

Step 4: Purify the product by high performance liquid chromatography to obtain a miR-210 mimic whose RNA sequence is 5'-U _S U _S GUGCGUGCGACAGCGAC _S U _S G _S A _S -Chol-3', the structure of which is shown in FIG. 1 .

experiment:

Experiment 1: MiR-210 mimics improve the immune activity of sea cucumbers against Vibrio splendidus infection Experiment 1. Transfection of miR-210 mimics

Mix 10 μL miR-210 mimic, 10 μL Lipofetamine 2000 and 80 μL PBS evenly as miR-210 transfection agent, 10 μL negative control, 10 μL Lipofetamine 2000 and 80 μL PBS as control, use a needle syringe (1 mL volume) from the body of sea cucumber On the side, the miR-210 mimic transfection agent and the control were injected into the body cavity of A. japonicus, which were the miR-210 transfection group and the control group, respectively.

2. Determination of Phagocytic Activity of Apostichopus coelomocytes

(1) One type of cells in the coelomocytes of sea cucumbers is phagocytic cells. The phagocytosis of phagocytic cells is the main strategy for cellular immunity of sea cucumbers. They are most active in identifying and resisting foreign non-self substances such as bacteria and viruses. The intensity of phagocytosis of phagocytic cells in the coelom fluid of ginseng can measure the level of immune response of A. japonicus. As shown in Figure 2, under normal circumstances, the phagocytes in the coelom fluid of A. japonicus are roughly round, with nuclei faintly visible, and can protrude pseudopodia into various shapes. The strength of phagocytic activity is indicated by the two indicators of phagocytic ability and phagocytic rate, in which the sum of the number of phagocytic cells approaching yeast cells with outstretched pseudopodia and the number of phagocytic cells that phagocytose yeast cells accounts for the total number of phagocytic cells The phagocytosis rate was expressed as the ratio of the number of phagocytic cells that phagocytized yeast cells to the total number of phagocytic cells.

(2) Preparation of yeast suspension: filter the settled seawater, filter it with a 0.45 μm cellulose acetate filter, and then autoclave to prepare sterile seawater; weigh 20 mg of edible dry yeast, add 200 mL of sterile seawater, and centrifuge at 3000 rpm. Discard the supernatant; add sterile seawater to resuspend, centrifuge, discard the supernatant to obtain a yeast suspension with a concentration of 2 mg/mL; add the autologous body cavity supernatant of Apostichopus japonicus and condition it at room temperature for more than 2 hours, centrifuge at 3000rpm, and discard the supernatant; Finally, 10 mL of yeast suspension was added, distributed into polyethylene centrifuge tubes, stored in a freezer at -20°C, and thawed before use.

(3) Calculation of phagocytosis ability and phagocytosis rate: 24 hours after transfection, the sea cucumbers of the miR-210 transfection group and the control group were soaked in a solution containing 1×10 ⁷ CFU/mL Vibrio brilliant (strain number D4501) In seawater, samples were taken at 4h and 24h after immersion, and 3 individuals (n=3) were randomly selected from each group at each sampling time point, and the body cavity fluid of each individual was collected separately for use. Mix 5 mL of body cavity fluid with an equal amount of conditioned yeast suspension, and draw 50 μL of the mixture at 30 minutes after the reaction to observe its phagocytosis and take pictures. Under the light microscope, the number of phagocytic cells in different states was recorded and the phagocytic ability and phagocytic rate were calculated. The calculation formula is:

Phagocytosis ability (%)=(total number of phagocytic cells extending pseudopodia close to yeast+total number of phagocytic cells that phagocytose yeast)/total number of phagocytic cells counted×100%;

Phagocytosis rate (%) = total number of phagocytic cells that phagocytized yeast/total number of counted phagocytic cells × 100%.

The results are shown in Figure 4 and Figure 5: at 4h and 24h after infection with Vibrio candidiasis, the results of the yeast phagocytosis experiment showed that compared with the control group transfected with negative control, the phagocytosis of sea cucumbers in the miR-210 transfection group The phagocytic ability and phagocytic rate of the cells were significantly enhanced (P<0.01). Among them, the phagocytic ability of phagocytic cells of A. japonicus in the miR-210 transfection group was increased by 52.82% compared with the control group, and the phagocytosis rate was increased by 47.69% compared with the control group at the 4th hour after infection with Vibrio splendidus (Fig. 4). At 24 hours after being infected with Vibrio splendidus, the phagocytic ability of phagocytic cells of A. japonicus in the miR-210 transfection group was increased by 36.24% compared with the control group, and the phagocytosis rate was increased by 32.45% compared with the control group (Figure 5) . Therefore, miR-210 mimics can be used as drugs to inhibit pathogenic bacteria of rot skin syndrome in cultured sea cucumbers.

Experiment 2: miR-210 mimetic enhances the immune activity of sea urchins against the infection of pathogenic Vibrio erythema (HD-1)

The method is the same as in Experiment 1, except that sea urchin intermedius was used instead of sea cucumber. Note that the injection was injected into the body cavity from the periosteal membrane of the oral surface of sea urchin intermedius. After 24 hours of transfection, miR-210 mimic transfection group and The sea urchins in the control group were all soaked in seawater containing 1×10 ⁴ CFU/mL Vibrio erythematosus HD-1 (NCBI accession number MH820372).

Figure 3 shows the morphology of the phagocytic cells of sea urchin intermedius and the process of phagocytosis of yeast cells. The results showed that, compared with the control group transfected with negative control, the phagocytic ability and phagocytic rate of phagocytic cells of sea urchins in the miR-210 transfection group were significantly enhanced (P<0.01). Among them, at the 4th hour of infection with Vibrio erythematosus, in the miR-210 mimic transfection group, the phagocytic ability of the phagocytic cells of sea urchin intermedius was enhanced by 24.16% compared with the control group, and the phagocytosis rate was higher than that of the control group. Group enhancement was 48.87% (Fig. 6). At 24 hours after infection with Vibrio erythematosus, the phagocytic ability of phagocytic cells of sea urchins in the miR-210 transfection group was increased by 24.15% compared with the control group, and the phagocytosis rate was increased by 32.09% compared with the control group (Figure 7). Therefore, the application of miR-210 mimics as a drug for inhibiting the pathogenic bacteria of erythema in cultured sea urchins.

Claims

A miR-210 mimic, characterized in that: the RNA sequence of the miR-210 mimic is 5'-U S U S GUGCGUGCGACAGCGAC S U S G S A S -Chol-3'.
A preparation method of miR-210 simulant as claimed in claim 1, characterized in that the following steps are carried out successively:

Step 1: using the RNA sequence of sea cucumber or sea urchin adult miR-210 to synthesize a single-stranded oligonucleotide, the RNA sequence of the adult miR-210 is shown in SEQ ID NO.1;

Step 2: Carrying out cholesterol modification on the 3' end of the oligonucleotide single strand;

Step 3: Carry out two thio-skeleton modifications to the 5' end of the oligonucleotide single-strand, perform four thio-skeleton modifications to the 3'-end of the oligonucleotide single-strand, and modify the oligonucleotide single-stranded whole chain carry out methoxy modification;

Step 4: Purify the product and obtain the RNA sequence as

miR-210 mimetic of 5′-U S U S GUGCGUGCGACAGCGAC S U S G S A S -Chol-3′.
An application of the miR-210 simulant described in claim 1 as an antibiotic substitute for breeding, characterized in that: it is used as a medicine for inhibiting putrefaction skin syndrome pathogenic bacteria for culturing sea cucumbers or as a drug for suppressing erythema pathogenic bacteria for culturing sea urchins. Application of bacteria drugs.