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Method for designing siRNA (Small Interfering Ribonucleic Acid) molecule resisting RNA virus

A technology of RNA virus and design method, applied in the field of RNA interference, can solve the problems of inefficiency, achieve a high degree of conservation, reduce the cost of anti-virus research and application, and facilitate the promotion and application

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

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Problems solved by technology

[0004] However, since the antiviral siRNA is only a short sequence of 21 base pairs, according to the conventional small RNA design principles, most viral genomes will produce hundreds of candidate siRNAs
Especially for RNA viruses with huge genomes like members of the Coronaviridae family, according to conventional design principles, it will take a lot of manpower and material resources to screen siRNAs that can effectively resist viruses in practical applications (due to reasons such as the spatial structure of the target RNA) , causing many siRNAs that theoretically meet the principle of "effectiveness" to be ineffective or inefficient in practical applications)

Method used

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  • Method for designing siRNA (Small Interfering Ribonucleic Acid) molecule resisting RNA virus
  • Method for designing siRNA (Small Interfering Ribonucleic Acid) molecule resisting RNA virus
  • Method for designing siRNA (Small Interfering Ribonucleic Acid) molecule resisting RNA virus

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Experimental program
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Effect test

Embodiment 1

[0021] Design and synthesis of small RNA molecules

[0022] (1) select the coding genes of TGEV structural protein and RdRP respectively;

[0023] (2) Using software to generate anti-TGEV siRNA molecules.

[0024] (3) According to the published small RNA sequence design rules, theoretically efficient siRNA molecules were selected and constructed into hairpin-type small RNA (shRNA) molecules (Table 1).

[0025] (4) The shRNA molecule was inserted into the vector to construct a plasmid expression vector containing U6 promoter.

Embodiment 2

[0027] Transfection of shRNA expression plasmids into ST cells

[0028] (1) ST cells (2-3×10 4 each / well) were inserted into a 48-well plate and cultured in complete DMEM;

[0029] (2) To the transfection reagent TransFast TM Add 400 μl DNase-free water to the transfection reagent, vortex to dissolve, and store at -20°C;

[0030] (3) 1.2 μg of dissolved plasmid DNA was dissolved in 300 μl of serum-free and double-antibody-free medium;

[0031] (4) Melt the transfection reagent at room temperature and vortex, add 3.6 μl to the DNA / DMEM mixture, vortex and mix; incubate at room temperature for 10-15 minutes to form a transfection complex;

[0032] (5) Remove the medium in the ST cells, add 100 μl / well of the transfection complex, and continue to store at 37°C in 5% CO 2 Incubate in the incubator for 1 hour;

[0033] Add 400 μl double antibody-free complete medium (containing 5% NBS) to ST cells; at 37°C, 5% CO 2 continue to grow in the incubator.

Embodiment 3

[0035] TGEV infection of ST cells

[0036] The shRNA expression plasmid was transfected into ST cells for 28 hours, then the cell culture medium was removed, and 200CCID was used 50 The amount of inoculation TGEV. After 40 hours, analyze cytopathic changes, cytotoxicity (MTS) or extract viral RNA for quantitative RT-PCR detection. .

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Abstract

The invention relates to a method for designing a siRNA (Small Interfering Ribonucleic Acid) molecule resisting RNA virus. The method comprises the following steps of: (1) selecting RNA polymerase, which is in RNA virus, has a conserved sequence and is RNA-dependent, as a target of siRNA; (2) utilizing siRNA design software to generate small RNA molecules resisting the RNA virus; (3) screening theoretically efficient siRNA for experiment research according to a siRNA design rule; and (4) adopting expression plasmid containing RNA polymerase III promoter U6 as an antiviral application form of the small RNA molecules. The method is applied to select an antiviral target sequence, so that the probability of obtaining efficient and antiviral small RNA molecules can be improved obviously, the selection range of an RNA interference target point is greatly reduced, the working efficiency is improved, and the cost for RNA interference technology research and application is obviously reduced.

Description

technical field [0001] The invention belongs to the technical field of RNA interference, in particular to a method for designing anti-RNA virus siRNA molecules. Background technique [0002] There are many types of RNA-like viruses, which are widely distributed, and are infected by aquatic and terrestrial animals as well as humans. For example, taura virus (TSV), Macrobrachium rosenbergii nodavirus (MrNV), infectious myonecrosis virus (IMNV), which pose a serious threat to aquatic cultured shrimp, and influenza virus (Flu virus), which is extremely harmful to terrestrial animals, including humans. ), human hepatitis A virus (HAV), human immunodeficiency virus (HIV), and various coronaviruses such as severe acute respiratory syndrome virus (SARS-CoV), porcine transmissible gastroenteritis virus (TGEV), etc. Due to the low activity of enzymes in the error repair mechanism of RNA viruses during RNA replication, there is almost no enzyme activity, so they mutate quickly; and va...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/63
Inventor 周俊芳华修国崔立房文红
Owner EAST CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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