Application of antisense nucleic acid amo-mir-9875 in preparation of anti-white spot syndrome virus preparation

A technology of amo-mir-9875 and antisense nucleic acid, which is applied in antiviral agents, applications, medical preparations containing active ingredients, etc., can solve the problems of affecting the ecological balance of breeding, unknown degradation difficulty, and large molecular weight of materials, etc. problem, to achieve the effect of small molecular weight, wide application range and quick effect

Active Publication Date: 2021-07-20
SHANTOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It requires a wide variety of raw materials, and the molecular weight of the materials contained is relatively large. The degree of degradation is unknown, and whether it will affect the ecological balance of breeding is also unknown.

Method used

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  • Application of antisense nucleic acid amo-mir-9875 in preparation of anti-white spot syndrome virus preparation
  • Application of antisense nucleic acid amo-mir-9875 in preparation of anti-white spot syndrome virus preparation
  • Application of antisense nucleic acid amo-mir-9875 in preparation of anti-white spot syndrome virus preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Detection test of miR-9875 expression level in Scylla pseudocarpus after WSSV infection

[0033] The blue crabs with a body weight of 30-40 g and a negative WSSV by qRT-PCR were domesticated in an indoor seawater tank at a salinity of 10‰ and a water temperature of 25°C for one week, and then injected 100 μL / crab on the fourth appendage, 10 6 Copy number of WSSV infection fluid. Scylla blood lymphocytes were collected at 0h, 24h and 48h after injection, total RNA was extracted and cDNA reverse transcription was carried out. The primers used were: 5-GTCGTATCCAGTGCAGGGTCCGAGGTCACTGGATACGACCTCCTCCC-3', and finally the expression of miR-9875 was detected by qPCR. The primers used were P1 and P2.

[0034] P1:5'-CGCCGGGAAGAGGAAGGG-3';

[0035] P2: 5'-TGCAGGGTCCGAGGTCACTG-3'.

[0036] see results figure 1 , the data showed that the expression of miR-9875 in the hemolymphocytes of Scylla pseudocarnata was significantly reduced after 24h and 48h of WSSV infection, indicating...

Embodiment 2

[0038] Overexpression test of miR-9875 in Scylla pseudocarpus

[0039]According to the method of Example 1, the crabs were randomly divided into 3 groups, and injected with 100 μL / miR-9875 mimic or miR-9875-scrambled or 100 μL / PBS control, respectively. 48 hours after the injection, the expression level of miR-9875 in blood lymphocytes of blue crabs was detected by referring to the method of Example 1.

[0040] see results figure 2 , the data showed that injection of miR-9875 mimics could significantly up-regulate the expression of miR-9875 in Scylla cryptae, which can be used for subsequent functional tests.

Embodiment 3

[0042] Experiment of miR-9875 promoting the replication of WSSV in Scylla burrowing crab

[0043] According to the method in Example 1, the mud crabs were randomly divided into 3 groups, the experimental group was injected with the mixture of WSSV and miR-9875, and the control group was injected with the mixture of WSSV and miR-9875-scrambled or PBS. The genomic DNA of the muscle tissue of the legs of Scylla pseudocavena was extracted respectively at 0h, 24h and 48h after injection, and then the virus copy number was detected by qPCR technology, and the primers used were P3 and P4.

[0044] P3: 5'-CAAATCTCCCCCTCATCTACTCAAC-3';

[0045] P4: 5'-AATAATTTTCCCGTTTCTGAATAGA-3'.

[0046] see results image 3 , the data showed that after the crabs were infected with WSSV, the injection of miR-9875 mimics could promote the replication of the virus in the host. Therefore, in order to screen anti-WSSV drugs, the antisense nucleic acid of miR-9875, AMO-miR- 9875 for antiviral tests.

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Abstract

The present invention relates to the application of antisense nucleic acid AMO-miR-9875 in the preparation of broad-spectrum anti-white spot syndrome virus preparations. The sequence of the small RNA antisense nucleic acid AMO-miR-9875 is: 5'-CUCCUCCCUUCCUCUUCC-3'. The small RNA antisense nucleic acid AMO-miR-9875 of the present invention can significantly inhibit the proliferation of WSSV in mud crabs, Penaeus japonicus and Procambarus clarkii, and greatly reduce the infection and lethal phenomenon of WSSV to seawater and freshwater cultured shrimp and crabs . The invention provides a potential drug for anti-white spot syndrome virus in aquaculture, which can efficiently and quickly inhibit the replication of WSSV, and has a wide range of applications, not only for the Penaeus japonicus and blue crabs that are cultured in seawater, but also for Procambarus clarkii cultured in freshwater. The small RNA antisense nucleic acid AMO-miR-9875 of the present invention has a small molecular weight and is easy to degrade, will not destroy the ecological balance of marine farming, and will not cause harm to the human body. It is an environmentally friendly and effective virus defense substance.

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 a small RNA antisense nucleic acid AMO-miR-9875 in the preparation of broad-spectrum anti-white spot syndrome virus preparations. Background technique [0002] Pseudomonas blue crab, referred to as blue crab, has the advantages of high nutritional value, delicious meat, fast growth, and strong adaptability. It is an important economically cultivated crab in my country. Penaeus japonicus, commonly known as flower prawns, monodon prawns, etc., is widely distributed, with large individuals, delicious meat, thick and hard carapace, resistance to dry dew, and resistance to capture. It is beneficial to long-distance transportation and has great economic value. Procambarus clarkii, also known as crayfish, is a freshwater shrimp, widely distributed in various water bodies, with fast growth, high reproduction rate, and strong adap...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K31/7088A61P31/20A23K50/80A23K20/153
CPCA61K31/7088A23K20/153A23K50/80A61P31/20
Inventor 龚燚李升康孔彤彤
Owner SHANTOU UNIV
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