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Application of miR-276 simulacrum to preparation of broad-spectrum white spot syndrome virus resisting medicines

A technology for mir-276 and white spot syndrome, applied in the field of virus prevention and control in aquaculture, can solve problems such as infection and no coping mechanism, achieve the effect of reducing shrimp death and wide application range

Active Publication Date: 2019-04-12
SHANTOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] The object of the present invention is to provide the application of miR-276 simulant in the preparation of broad-spectrum anti-white spot syndrome virus medicine, to solve the increasingly high degree of intensification of aquaculture at the present stage, WSSV, as a kind of pantropic virus, many Aquaculture species are often infected by WSSV at the same time, but there is no mature coping mechanism at this stage

Method used

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  • Application of miR-276 simulacrum to preparation of broad-spectrum white spot syndrome virus resisting medicines
  • Application of miR-276 simulacrum to preparation of broad-spectrum white spot syndrome virus resisting medicines
  • Application of miR-276 simulacrum to preparation of broad-spectrum white spot syndrome virus resisting medicines

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Experimental program
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Effect test

Embodiment 1

[0027] Screening of key miRNAs against WSSV infection in Penaeus japonicus

[0028] Japonica shrimp with a body weight of about 10 g was temporarily raised at 25°C for 3 days, and then 50 μL / piece of PBS buffer solution or WSSV infection solution was injected into the 2-3 abdominal segment muscle of the shrimp with a 1 ml syringe. Shrimp blood cells were collected at 0h, 12h, and 24h after injection for miRNA sequencing.

[0029] WSSV was injected into the experimental group Penaeus japonicus, and the control group was injected with PBS. The hemolymph of the experimental group and the control group were collected after 12h and 24h, respectively, and microRNA sequencing was performed after treatment.

[0030] see results figure 1 , the results showed that the expression of miR-276 in Penaeus japonicus was significantly increased after 12h and 24h of WSSV infection, indicating that miR-276 was involved in the host's immune response to the virus and was a potential antiviral tar...

Embodiment 2

[0032] Experiment of miR-276 mimics inhibiting the proliferation of WSSV in Penaeus japonicus

[0033] Penaeus japonica was injected with 50 μL / WSSV infection solution, followed by 50 μg / synthetic miR-276 mimic (5'-TAGGAACTTCATACCGTGCTCT-3'), and the control group was scrambled miR-276 nucleotides (miR-276 -scrambled: 5'-GTTTCTTCAAGTCACACACTGTG-3'). After 0h, 24h, 36h and 48h of WSSV infection, appropriate amount of back muscle of shrimp was taken, and then TAKARA's Premix Ex TaqTM (Probe qPCR) kit was used to detect the WSSV target gene fragment, and the detection primers were P1 and P2 respectively.

[0034] P1: 5'-ATGTCTGCATCTTTAAT-3'; P2: 5'-TTATGACACAAACCTAT-3'.

[0035] see results figure 2 , 36h and 48h after injection of miR-276 mimics, the copy number of WSSV in Penaeus japonicus was significantly lower than that of the control group injected with miR-276-scrambled, indicating that miR-276 mimics significantly inhibited the replication of WSSV in Penaeus japonicus ...

Embodiment 3

[0037] MiR-276 mimetic reduces cumulative mortality of Penaeus japonicus after WSSV infection

[0038]Japanese prawns with a body weight of about 10 g were temporarily cultured at 25°C for 3 days and then divided into 5 groups: no treatment group, PBS injection group, WSSV injection group, WSSV+miR-276 injection group and WSSV+miR-276-scrambled injection group. Group 20. See Example 1 for the injection method, observe 3 times a day for 5 days, clean up dead shrimp in time and record the number of deaths.

[0039] see results image 3 , within five days after injection, the mortality rate of the WSSV-injected group and the WSSV+miR-276-scrambled-injected group gradually increased, reaching almost 100% on the fifth day. In contrast, the mortality of Penaeus japonicus in the WSSV+miR-276 injection group was significantly reduced, indicating that after WSSV infection of Penaeus japonicus, injection of miR-276 mimics can significantly reduce the death of shrimp caused by WSSV inf...

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Abstract

The invention relates to an application of miR-276 simulacrum to preparation of broad-spectrum white spot syndrome virus resisting medicines. The miR-276 simulacrum is artificially-synthesized penaeusjaponicus miR-276, and the sequence is 5'-TAGGAACTTCATACCGTGCTCT-3'. The content of the miR-276 after the penaeus japonicus is infected with WSSV significantly rises; reproduction of the WSSV in bodies of the penaeus japonicus can be significantly restrained, the virus resisting capacity of the penaeus japonicus can be improved, and the survival rate of the penaeus japonicus can be significantlypromoted; and the reproduction of the WSSV in bodies of procambarus clarkii and bodies of scylla paramamosain can be restrained. The miR-276 simulacrum can be used as a new medicine for efficiently preventing infection and propagation of the WSSV, is wide in application range, is suitable for breeding the penaeus japonicus and the scylla paramamosain in sea, is also suitable for breeding procambarus clarkii in fresh water, does not influence food safety, and contributes to long-term sustainable development of the aquaculture industry.

Description

technical field [0001] The invention belongs to the field of aquaculture virus prevention and control, and in particular relates to a miR-276 simulant and its application in preparing broad-spectrum anti-white spot syndrome virus medicine. Background technique [0002] Penaeus japonicus is widely distributed in the world, and it is mainly distributed in the coastal area south of the Yangtze River in my country. Its meat is tender and nutritious, and it is an important breeding object in many countries and regions. In the 1980s, the development of my country's shrimp farming industry led to the rapid development of my country's aquaculture industry and promoted the economic growth of my country's coastal areas. However, in the early 1990s, shrimp viral infections began to spread in the farms, resulting in The economic benefits of the shrimp farming industry plummeted. In recent years, due to the influence of factors such as environmental pollution and excessive breeding dens...

Claims

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Application Information

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IPC IPC(8): C12N15/113A61K31/7088A61P31/14A23K20/153A23K50/80
CPCA61P31/14A23K20/153A23K50/80A61K31/7088C12N15/113C12N2310/141
Inventor 龚燚陈娇李升康
Owner SHANTOU UNIV