CN113201544A - miR-210 simulant, preparation method and application of miR-210 simulant as antibiotic substitute for breeding - Google Patents
miR-210 simulant, preparation method and application of miR-210 simulant as antibiotic substitute for breeding Download PDFInfo
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Abstract
The invention discloses a miR-210 mimic, wherein the RNA sequence is 5' -USUSGUGCGUGCGACAGCGACSUSGSAS-Chol-3'. The preparation method comprises the following steps in sequence: synthesizing an oligonucleotide single strand by using an RNA sequence of an adult miR-210, wherein the RNA sequence of the adult miR-210 is shown as SEQ ID NO. 1; carrying out cholesterol modification on the 3' end of the oligonucleotide single strand; doubling the 5' end of the oligonucleotide single strandModifying a single-thio-skeleton, namely performing four-thio-skeleton modification on the 3' end of the oligonucleotide single-chain, and performing methoxy modification on the whole oligonucleotide single-chain; purifying the product to obtain the miR-210 analogue with the RNA sequence shown as SEQ ID NO. 1. The miR-210 simulant can be used as a medicament for inhibiting pathogenic bacteria of the rotten skin syndrome for cultivating stichopus japonicus or a medicament for inhibiting pathogenic bacteria of the red spot for cultivating sea urchins.
Description
Technical Field
The invention relates to the field of aquatic animal disease prevention and treatment technologies and development of environment-friendly antibiotic drug substitutes, and particularly relates to a miR-210 mimic, a preparation method and application of the miR-210 mimic as an antibiotic substitute for cultivation.
Background
The aquaculture diseases caused by pathogenic bacteria are the core problems restricting the development of the aquaculture industry in China, and the current main coping strategy is to use various antibiotic medicines. The antibiotic medicines are not used in a standard way, so that the aquaculture pathogenic bacteria with strong drug resistance and multiple drug resistance are generated, and the antibiotic medicines have the characteristics of higher and higher dosage, more and more types of administration and the like in aquaculture. Research shows that only about 20% of antibiotics put into a water body in the aquaculture industry are utilized by organisms, residual antibiotics exist in the water body for a long time, deep and complex influences can be generated on the water body quality, the ecological diversity of the water body, the distribution and abundance of organism populations and the like, in addition, the antibiotics used in the aquaculture industry at present are all human and animal shared medicines, and the antibiotic residues can also generate potential threats to the human health. Therefore, the development of environment-friendly and efficient antibiotic drug substitutes is one of the urgent needs for promoting the green and healthy aquaculture at present.
microRNAs (miRNAs) are a kind of endogenous small molecule non-coding RNA (non-coding RNA) composed of 21-25 nucleotides, which are commonly present in animals and plants, and can degrade miRNA or inhibit the translation of related proteins by combining with seed Regions (seed Regions) in the 3' non-coding sequences (3 ' Untrancoded Regions, 3' UTR) of target genes, thereby regulating the expression of the target genes at the post-transcriptional level. miRNAs mimics are artificially synthesized small molecular compounds, and regulate the biological function of a target gene by simulating endogenous miRNA of an organism, so that the miRNAs play an important role in regulating and controlling various vital activities in the organism and have no influence on the environment and human health. In the disease control research of aquatic animals, miRNAs mimics are used as antibiotic substitutes, so that the research of the miRNAs mimics applied to the development of green, healthy and pollution-free economic aquatic animal antibacterial drugs is not reported.
Disclosure of Invention
The invention aims to solve the technical problems in the prior art and provides a miR-210 mimic, a preparation method and application of the miR-210 mimic as an antibiotic substitute for breeding.
The technical solution of the invention is as follows: a miR-210 mimetic, comprising: the RNA sequence of the miR-210 mimic is 5' -USUSGUGCGUGCGACAGCGACSUSGSAS-Chol-3'。
The preparation method of the miR-210 mimic sequentially comprises the following steps:
step 1: synthesizing an oligonucleotide single strand by utilizing an RNA sequence of an adult miR-210 of stichopus japonicus or sea urchin, wherein the RNA sequence of the adult miR-210 is shown as SEQ ID NO. 1;
step 2: carrying out cholesterol modification on the 3' end of the oligonucleotide single strand;
and step 3: carrying out two thio-skeleton modifications on the 5 'end of the oligonucleotide single strand, carrying out four thio-skeleton modifications on the 3' end of the oligonucleotide single strand, and carrying out methoxy modification on the whole oligonucleotide single strand;
and 4, step 4: purifying the product to obtain RNA with 5' -U sequenceSUSGUGCGUGCGACAGCGACSUSGSAS-miR-210 mimetic of Chol-3'.
The application of the miR-210 simulant as an antibiotic substitute for cultivation is the application of a medicament for inhibiting pathogenic bacteria of the pythium aphanidermatum syndrome for cultivating stichopus japonicus or the application of a medicament for inhibiting pathogenic bacteria of the red spot disease for cultivating sea urchins.
The miR-210 simulant is prepared from the miR-210 sequence of the adult stichopus japonicus or echinococcus intermedius, and has the advantages of simple in-vitro synthesis and convenient use. In particular, the miR-210 adopted has a sequence which is different from that of miR-210 in other species including human beings, does not interfere normal gene expression of other organisms, and has no influence on ecological diversity and human health. The miR-210 mimics are respectively transfected into coelomic fluids of the stichopus japonicus and the sea urchins, so that the phagocytic activity of the stichopus japonicus and the sea urchins can be remarkably improved, the miR-210 mimics can replace antibiotics to play the function of antibacterial drugs, and various defects caused by the use of the antibiotics are avoided.
Drawings
FIG. 1 is a schematic diagram of the sequence structure of a miR-210 mimic according to an embodiment of the invention.
FIG. 2 is a diagram of the morphology of phagocytic cells and the phagocytosis process of Stichopus japonicus.
FIG. 3 is a schematic diagram of the experimental results of the effect of miR-210 mimics transfected after infection of the pathogenic bacteria Vibrio splendidus on the phagocytic capacity of stichopus japonicus phagocytic cells.
FIG. 4 is a schematic diagram of experimental results of the influence of transfected miR-210 mimics after infection of the pathogenic bacteria Vibrio splendidus on phagocytic rate of stichopus japonicus.
FIG. 5 is a diagram of the phagocytic morphology and phagocytosis process of Strongylocentrotus intermedius.
FIG. 6 is a schematic diagram showing the experimental results of the effect of miR-210 mimics transfected after infecting red spot pathogenic vibrio on the phagocytic capacity of echinococcus intermedius phagocytes.
FIG. 7 is a schematic diagram showing the experimental results of the effect of miR-210 mimics transfected after infecting red spot pathogenic vibrio on the phagocytic rate of echinococcus intermedius phagocytes.
Detailed Description
The preparation method of the miR-210 simulant is sequentially carried out according to the following steps:
step 1: synthesizing an oligonucleotide single strand by utilizing an RNA sequence of an adult miR-210 of stichopus japonicus or sea urchin, wherein the RNA sequence of the adult miR-210 is shown as SEQ ID NO. 1;
step 2: carrying out cholesterol modification on the 3' end of the oligonucleotide single strand;
and step 3: carrying out two thio-skeleton modifications on the 5 'end of the oligonucleotide single strand, carrying out four thio-skeleton modifications on the 3' end of the oligonucleotide single strand, and carrying out methoxy modification on the whole oligonucleotide single strand;
and 4, step 4: purifying the product by high performance liquid chromatography to obtain RNA with 5' -U sequenceSUSGUGCGUGCGACAGCGACSUSGSASmiR-210 mimics of Chol-3', the structure of which is shown in FIG. 1.
Experiment:
experiment 1: miR-210 simulant for improving resistance of stichopus japonicus to vibrio splendidus (Vibrio splendidus)Vibrio splendidus) Immune activity assay of infection
1. Transfection of miR-210 mimetics
And uniformly mixing 10 mu L of miR-210 simulant, 10 mu L of Lipofetamine 2000 and 80 mu L of PBS to serve as miR-210 transfection agent, uniformly mixing 10 mu L of negative control, 10 mu L of Lipofetamine 2000 and 80 mu L of PBS to serve as control, and injecting the miR-210 simulant transfection agent and the control into the body cavity of the stichopus japonicus from the side surface of the body of the stichopus japonicus by using a needle syringe (1 mL range), wherein the miR-210 transfection agent and the control are respectively a miR-210 transfection group and a control group.
2. Determination of body cavity cell phagocytic activity of stichopus japonicus
(1) One type of cells in the body cavity cells of the stichopus japonicus are phagocytic cells, the phagocytosis of the phagocytic cells is a main strategy for cellular immunity of the stichopus japonicus, and the phagocytic effect is most active in identifying and resisting bacteria, viruses and other foreign non-self substances, and the level of the immune response of the stichopus japonicus is usually measured by the strength of the phagocytic effect of the phagocytic cells in the body cavity liquid of the stichopus japonicus. As shown in FIG. 2, phagocytes in the body cavity fluid of Apostichopus japonicus are normally approximately round, so that the nucleus is hidden and visible, and pseudopodia can be stretched out to change into various shapes. The strength of phagocytic activity is indicated by two indexes, i.e., phagocytic ability expressed by the ratio of the total number of phagocytes which begin to protrude pseudopodia toward the yeast cells to the total number of phagocytes which phagocytes the yeast cells to the total number of phagocytes, and phagocytic rate expressed by the ratio of the number of phagocytes which phagocytes the yeast cells to the total number of phagocytes.
(2) Preparation of yeast suspension: filtering the filtered and settled seawater with 0.45 μm acetate fiber filter membrane, and autoclaving to obtain sterile seawater; weighing 20 mg of edible dry yeast, adding 200 mL of sterile seawater, centrifuging at 3000 rpm, and discarding the supernatant; adding sterile seawater for resuspension, centrifuging, and discarding the supernatant to obtain a yeast suspension with the concentration of 2 mg/mL; adding the supernatant of the body cavity of the stichopus japonicus, regulating the temperature at room temperature for more than 2 h, centrifuging at 3000 rpm, and discarding the supernatant; finally, 10 mL of yeast suspension is added, subpackaged in polyethylene centrifuge tubes, frozen and stored at-20 ℃, and thawed before use.
(3) Calculation of phagocytic capacity and phagocytic rate: after 24 h of transfection, the stichopus japonicus of the miR-210 transfection group and the stichopus japonicus of the control group are soaked in a solution containing 1 × 107Sampling in seawater of CFU/mL vibrio splendidus (strain number is D4501) at 4 h and 24 h after soaking, randomly selecting 3 individuals (n = 3) from each group at each sampling time point, and collecting coelomic fluid of each individual for later use. 5 mL of coelomic fluid was mixed with an equal amount of conditioned yeast suspension, and 50. mu.L of the mixture was withdrawn at 30 min after the reaction to observe phagocytosis and photographed. Under a light microscope, the number of phagocytic cells in different states is recorded respectively, and the phagocytic capacity and the phagocytic rate are calculated, wherein the calculation formula is as follows:
phagocytic capacity (%) - (total number of phagocytic cells protruding pseudopodium-proximal to yeast + total number of phagocytic cells phagocytizing yeast)/total number of phagocytic cells counted × 100%;
the phagocytosis ratio (%) -% is the total number of phagocytic cells phagocytizing yeast/the total number of phagocytic cells counted × 100%.
The results are shown in FIGS. 4 and 5: the results of the yeast phagocytosis experiments at 4 h and 24 h infected with Vibrio splendidus show that the phagocytic capacity and phagocytic rate of the phagocytic cells of the stichopus japonicus in the miR-210 transfected group are remarkably enhanced compared with those of the control group transfected with negative control (the phagocytic capacity and the phagocytic rate of the phagocytic cells of the stichopus japonicus in the miR-210 transfected group are remarkably enhanced: (P <0.01). Wherein, at the 4 h of Vibrio splendidus infection, the phagocytic capacity of the phagocytic cells of the stichopus japonicus in the miR-210 transfection group is increased by 52.82% compared with that of the control group, and the phagocytic rate is increased by 47.69% compared with that of the control group (figure 4). At the 24 h of Vibrio splendidus infection, the phagocytic capacity of the phagocytic cells of the stichopus japonicus in the miR-210 transfected group is increased by 36.24% compared with that of the control group, and the phagocytic rate is increased by 32.45% compared with that of the control group (figure 5). Therefore, the miR-210 simulant can be used as a medicament for inhibiting pathogenic bacteria of the skin rot syndrome in the culture of the stichopus japonicus.
Experiment 2: experiment for improving immune activity of strongylocentrotus intermedius against infection of vibrio ceriferus (HD-1) by miR-210 mimics
The method is the same as experiment 1, except that sea urchin medicinalis is used to replace stichopus japonicus, the sea urchin medicinalis is injected into a body cavity from the peristomal membrane of the oral surface of the sea urchin medicinalis during injection, and after transfection is carried out for 24 h, the sea urchin medicinalis of miR-210 simulant transfection group and control group are soaked in a solution containing 1 × 104CFU/mL seawater of Vibrio cerulosa HD-1 (NCBI accession No. MH 820372).
The phagocytic morphology of echinococcus intermedius and the process of phagocytizing yeast cells are shown in FIG. 3, and the results of the phagocytic activity assay are shown in FIGS. 6 and 7: the results of the yeast phagocytosis experiments at 4 h and 24 h of infection with pathogenic vibrio erythroseptoria show that the phagocytic capacity and phagocytic rate of the phagocytic cells of the echinococcus intermedius in the miR-210 transfected group are remarkably enhanced compared with those of the control group transfected with negative control (the)P <0.01). Wherein, at the 4 h of infecting red spot pathogenic vibrio, the phagocytic capacity of the phagocytic cells of the echinococcus intermedium in the miR-210 mimic transfection group is enhanced by 24.16% compared with the control group, and the phagocytic rate is enhanced by 48.87% compared with the control group (FIG. 6). At the 24 h of infection with vibrio rubellus, the phagocytic capacity of the phagocytic cells of echinococcus intermedia in the miR-210 transfected group was increased by 24.15% compared with the control group, and the phagocytic rate was increased by 32.09% compared with the control group (fig. 7). Therefore, the miR-210 simulant is applied to the medicines for inhibiting the pathogenic bacteria of the red spot disease for culturing the sea urchins.
Sequence listing
<110> university of Dalian ocean
<120> miR-210 simulant, preparation method and application thereof as antibiotic substitute for breeding
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> RNA
<213> microRNAs（miRNAs）
<400> 1
uugugcgugc gacagcgacu ga 22
Claims (3)
1. A miR-210 mimetic, comprising: the RNA sequence of the miR-210 mimic is 5' -USUSGUGCGUGCGACAGCGACSUSGSAS-Chol-3'。
2. A method for preparing the miR-210 mimic according to claim 1, wherein the method comprises the following steps in sequence:
step 1: synthesizing an oligonucleotide single strand by utilizing an RNA sequence of an adult miR-210 of stichopus japonicus or sea urchin, wherein the RNA sequence of the adult miR-210 is shown as SEQ ID NO. 1;
step 2: carrying out cholesterol modification on the 3' end of the oligonucleotide single strand;
and step 3: carrying out two thio-skeleton modifications on the 5 'end of the oligonucleotide single strand, carrying out four thio-skeleton modifications on the 3' end of the oligonucleotide single strand, and carrying out methoxy modification on the whole oligonucleotide single strand;
and 4, step 4: purifying the product to obtain an RNA sequence of
5'-USUSGUGCGUGCGACAGCGACSUSGSAS-miR-210 mimetic of Chol-3'.
3. The use of the miR-210 mimetic of claim 1 as a replacement for antibiotics for aquaculture, wherein: the application of the compound is used as a medicament for inhibiting pathogenic bacteria of the skin rot syndrome for cultivating stichopus japonicus or a medicament for inhibiting pathogenic bacteria of the red spot disease for cultivating sea urchins.
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| PCT/CN2021/104504 WO2022241922A1 (en) | 2021-05-20 | 2021-07-05 | Mir-210 mimic, and preparation method therefor and use thereof as antibiotic substitute for culture |
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| CN102080083A (en) * | 2009-12-01 | 2011-06-01 | 中国科学院上海药物研究所 | Human miR-149 antisense nucleotide and application thereof |
| CN102382825A (en) * | 2010-09-01 | 2012-03-21 | 中国科学院上海药物研究所 | Human miR-1826 antisense nucleic acid and application thereof |
| CN106167824A (en) * | 2016-07-14 | 2016-11-30 | 西北工业大学 | Application with the oligonucleotide compound of senile osteoporosis related microRNA |
| CN110484537A (en) * | 2019-09-02 | 2019-11-22 | 中国水产科学研究院淡水渔业研究中心 | A kind of preparation method and application of miR-92 promotor and its injection |
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- 2021-07-05 WO PCT/CN2021/104504 patent/WO2022241922A1/en active Application Filing
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| CN102080083A (en) * | 2009-12-01 | 2011-06-01 | 中国科学院上海药物研究所 | Human miR-149 antisense nucleotide and application thereof |
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| CN106167824A (en) * | 2016-07-14 | 2016-11-30 | 西北工业大学 | Application with the oligonucleotide compound of senile osteoporosis related microRNA |
| CN110484537A (en) * | 2019-09-02 | 2019-11-22 | 中国水产科学研究院淡水渔业研究中心 | A kind of preparation method and application of miR-92 promotor and its injection |
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