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
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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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