Target spot selecting method for efficiently inhibiting WSSV (White Spot Syndrome Virus)

An efficient and targeted technology, applied in the field of RNA interference and anti-virus, can solve the problems of unfavorable promotion and application of RNA interference technology, high price of small RNA molecule synthesis, and increase of research cost, so as to facilitate promotion and application, narrow the scope of selection, The effect of reducing research costs

Inactive Publication Date: 2012-08-22
EAST CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Due to the high price of small RNA molecule synthesis and the long process of animal screening research, it is bound to be time-consuming and labor-intensive to screen real and efficient small ...

Method used

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  • Target spot selecting method for efficiently inhibiting WSSV (White Spot Syndrome Virus)
  • Target spot selecting method for efficiently inhibiting WSSV (White Spot Syndrome Virus)
  • Target spot selecting method for efficiently inhibiting WSSV (White Spot Syndrome Virus)

Examples

Experimental program
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Effect test

Embodiment 1

[0018] Design and synthesis of siRNA molecules

[0019] (1) Select the coding sequences of enzymes related to WSSV replication and metabolism, respectively.

[0020] (2) Using software to generate small RNA interference molecules against WSSV.

[0021] (3) According to the published small RNA sequence design rules, select a theoretically efficient siRNA, see Table 1.

[0022] (4) The above small RNA molecules are constructed into a hairpin structure, that is, shRNA molecules.

[0023] Table 1

[0024]

Embodiment 2

[0026] siRNA molecule anti-WSSV test (RNA interference test)

[0027] Healthy Litopenaeus vannamei with a body weight of 7-9g and a negative PCR test were temporarily raised in an indoor pond for 1 week, and were randomly divided into different siRNA interference groups, one positive control group and one negative control group, and they were temporarily raised in 90L white glass fiber in the barrel. Insulin syringes were used to inject 50 μL / siRNA (25 μg) intramuscularly into one side of the fourth abdominal segment of Litopenaeus vannamei in the interference group, and 50 μL / WSSV infection solution was injected into the contralateral muscle 4 hours later; Inject 50 μL / mouse of 0.01mol / L PBS (pH 7.4) and 50 μL / mouse of WSSV infection solution, while the negative control group was injected with 50 μL / mouse of 0.01mol / L PBS (pH 7.4) twice. The water temperature for shrimp culture is controlled at 27-28°C, 1 / 2 of the water is changed every day, and the aeration is continued for...

Embodiment 3

[0029] Study on Effects of Various siRNAs on Shrimp Mortality

[0030] The prawns were randomly divided into 5 siRNA (RR9, RR1, DPI, DP2 and PK1) interference groups, 1 positive control group (WSSV), and 1 negative control group (Negative), with 9 prawns in each group. The RNAi experiment was carried out according to the method in Example 2. Observe 4 times a day, pick out dead and dying prawns in time, record the number of deaths, and detect dead prawns with WSSV-specific PCR primers, continue to observe for 7 days, and repeat the experiment 3 times. figure 1 It is a comparison chart of the effects of different siRNAs on the mortality of prawns.

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Abstract

The invention relates to a target spot selecting method for efficiently inhibiting WSSV (White Spot Syndrome Virus), which comprises the steps of: 1, selecting coded sequences of ribonucleotide reductase and DNA polymerase in a WSSV genome; 2, generating a WSSV-resistant small RNA interference molecule by using software; and 3, screening theoretically efficient WSSV-resistant siRNA (small interfering RNA) according to a siRNA design rule. The small RNA molecule is designed by using the target spot selecting method, thus the selection range of the virus-resistant small RNA molecule can be effectively narrowed, the working efficiency is increased, the research cost is remarkably lowered, and the popularization and the application of an RNA interference technology in the aquatic virus-resistant field are facilitated.

Description

technical field [0001] The invention belongs to the field of RNA interference anti-virus, and in particular relates to a target selection method for efficiently inhibiting WSSV. Background technique [0002] As the pathogen of white spot disease (WSD) in prawns, white spot syndrome virus (WSSV) ranks among the "needs" due to its pan-tropicity, high pathogenicity and high lethality to the host. The first of the three major viral pathogens of prawns reported by OIE. Typical symptoms of prawns—red bodies and white spots on the carapace—can appear after 48 hours of infection, and the mortality rate can be as high as 100% within 3 to 10 days. Therefore, since the disease broke out for the first time in 1993, it has brought huge economic losses to the shrimp farming industry in my country almost every year, and the phenomenon of no harvest at the mouth of the pond often occurs. Like other invertebrates, the lack of specific immune mechanism in shrimp greatly increases the diffic...

Claims

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Application Information

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IPC IPC(8): G06F19/16
Inventor 周俊芳
Owner EAST CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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