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Application of antisense nucleic acid AMO-miR-307 to preparation of preparation for resisting white spot syndrome viruses

A technology for amo-mir-307 and white spot syndrome, applied in antiviral agents, DNA/RNA fragments, recombinant DNA technology, etc., can solve problems such as health impacts, antibiotics destroying the ecological environment of breeding areas, and lack of prevention and control methods, and achieve Quick effect, wide application range, and easy degradation of molecular weight

Active Publication Date: 2019-07-23
SHANTOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Antibiotics have been widely used in aquaculture for a long time. Although they can inhibit the invasion of viruses and bacteria to a certain extent, in the long run, the abuse of antibiotics will greatly damage the ecological environment of the farming area, and their accumulation in the human body will also be harmful to health. Make an impact is now banned from using
At present, the prevention and control of immune diseases of invertebrates has made great progress, but there is still a lack of rapid and effective prevention and control methods. Seeking environmentally friendly and efficient WSSV prevention and control methods has far-reaching significance for the long-term development of the aquaculture industry.

Method used

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  • Application of antisense nucleic acid AMO-miR-307 to preparation of preparation for resisting white spot syndrome viruses
  • Application of antisense nucleic acid AMO-miR-307 to preparation of preparation for resisting white spot syndrome viruses
  • Application of antisense nucleic acid AMO-miR-307 to preparation of preparation for resisting white spot syndrome viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Detection test of miR-307 expression level in Scylla pseudophylla after WSSV infection

[0032] Select 30-40g of blue crabs to domesticate for a week in seawater with a salinity of 10-12‰, and keep the water temperature at about 22°C. Use a 1ml syringe to inject 200 μL / only at the joint of the fourth appendage of the mud crab, 10 6 WSSV dilution of copy number, collect blood lymphocytes at 0h, 24h and 48h after injection, use RNA extraction kit to extract RNA and then use cDNA reverse transcription kit to synthesize cDNA, the reverse transcription primer used is 5'-GTCGTATCCAGTGCAGGGTCCGAGGTCACTGGATACGACCTCACTCA -3', finally detect the expression level of miR-307 by qRT-PCR, the primers used are P1 and P2.

[0033] P1:5'-CGCCGTCACAACCTCCTTGAGTGAG-3';

[0034] P2: 5'-TGCAGGGTCCGAGGTCACTG-3'.

[0035] see results figure 1 , qRT-PCR data showed that after WSSV infection, the expression of miR-307 in the hemolymph of mud crabs was significantly down-regulated, indicatin...

Embodiment 2

[0037] Experiment of miR-307 mimics up-regulating the expression of miR-307 in Scylla macula

[0038]According to the method of Example 1, the mud crabs were cultivated and randomly divided into three groups. The experimental group was injected with 100 μL / miR-307 mimic (10 nM) synthesized in vitro, and the control group was injected with 100 μL / miR-307- scrambled (miR-307 mimic scrambled sequence) 10 nM or 100 μL / PBS, 48 hours after injection, blood lymphocytes were collected, and the expression level of miR-307 in the mud crab was detected according to the method of Example 1.

[0039] see results figure 2 , the data show that injection of miR-307 mimics can significantly up-regulate the expression of miR-307 in mud crabs, which can be used for subsequent functional tests.

Embodiment 3

[0041] MiR-307 mimics promote the proliferation of WSSV in Scylla burrowing crabs

[0042] According to the method in Example 2, the mud crabs were randomly divided into 3 groups, the experimental group was injected with the mixture of WSSV infection solution and miR-307, and the negative control group was injected with the mixture of WSSV infection solution and miR-307-scrambled or only injected WSSV infection solution. 0h, 24h, and 48h after injection, an appropriate amount of muscle tissue from the legs of Scylla pseudocavena was taken, and the total DNA was extracted using a genomic DNA extraction kit (Tiangen), and then the virus copy number was detected by qRT-PCR with primers P3 and P4.

[0043] P3: 5'-CAAATCTCCCCCTCATCTACTCAAC-3';

[0044] P4: 5'-AATAATTTTCCCGTTTCTGAATAGA-3'.

[0045] see results image 3 , 24h and 48h after injection of miR-307 mimics, the copy number of WSSV in Scylla pseudocarpus was significantly more than that in the control group, indicating t...

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Abstract

The invention relates to an application of antisense nucleic acid AMO-miR-307 to preparation of a preparation for resisting white spot syndrome viruses. The sequence of the antisense nucleic acid AMO-miR-307 is 5'-CUCACUCAAGGAGGUUGUGA-3'. The antisense nucleic acid AMO-miR-307 has notable resisting effects on WSSV. Host hemolymph cells are promoted to be in apoptosis so as to restrain the proliferation of viruses, and effectively restrain proliferation of WSSV in scylla paramamosain, marsupenaeus japonicus and procambarus clarkii. The antisense nucleic acid AMO-miR-307 provides a potential medicine for preventing and treating the white spot syndrome viruses, so that the infection and the propagation of the white spot syndrome viruses can be effectively prevented and treated, and the antisense nucleic acid AMO-miR-307 is broad in application range, is suitable for marsupenaeus japonicus and the scylla paramamosain which are bred in seawater, and is also suitable for the procambarus clarkii bred in fresh water. The antisense nucleic acid AMO-miR-307 is green and environmental-friendly, cannot influence ecological balance in breeding process, and cannot cause damage to health of humanbodies.

Description

technical field [0001] The invention relates to the field of prevention and treatment of viral diseases in aquaculture, in particular to the application of an antisense nucleic acid AMO-miR-307 in the preparation of anti-white spot syndrome virus preparations. Background technique [0002] The blue crab is an important marine economic cultured crab with extremely high economic value. There are abundant crab resources in the ocean. There are more than 2,000 species of crabs in the Indian Ocean and the Pacific Ocean alone. However, there are very few crabs that can be cultured artificially. Among them, blue crabs occupy a very important position and are mainly distributed in my country. in the southeast coast and Taiwan. Penaeus japonicus, belonging to Crustacea, is loved by farmers because of its strong stress resistance and disease resistance, and its high economic value, and it is widely cultivated in the southeast coast of my country. Procambarus clarkii, commonly known a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K31/7105A61P31/12
CPCC12N15/1131A61K31/7105A61P31/12C12N2310/141Y02A40/81
Inventor 龚燚李升康任鑫
Owner SHANTOU UNIV
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