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
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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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