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siRNA (small interfering ribonucleic acid) capable of inhibiting expression of enterovirus 71 type gene, composition and application

A technology of enteroviruses and compositions, applied in the direction of DNA/RNA fragments, gene therapy, antiviral agents, etc., can solve the problems of high toxicity and side effects, unclear, low specificity, etc., and achieve the effect of high inhibition rate

Active Publication Date: 2013-05-01
SUZHOU RIBO LIFE SCIENCE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there is no specific treatment method, but mainly supportive therapy. The main drugs used are interferon and other spectrum antiviral drugs (acyclovir, ganciclovir, ribavirin, etc.), but the disadvantages of these drugs Poor pertinence, low specificity, high toxicity or unclear side effects

Method used

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  • siRNA (small interfering ribonucleic acid) capable of inhibiting expression of enterovirus 71 type gene, composition and application
  • siRNA (small interfering ribonucleic acid) capable of inhibiting expression of enterovirus 71 type gene, composition and application
  • siRNA (small interfering ribonucleic acid) capable of inhibiting expression of enterovirus 71 type gene, composition and application

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Experimental program
Comparison scheme
Effect test

preparation example Construction

[0209] The preparation method of siRNA provided by the invention includes the design of siRNA sequence and the synthesis of siRNA sequence.

[0210] Select the whole genome sequence of EV71 isolated in various regions of my country in recent years for gene comparison, select the highly conserved EV71 virus genome (Genbank accession number is FJ713137) as the template, and then select the 19bp core for the conserved region of the EV71 virus gene respectively. Nucleotide sequence, design siRNA.

[0211] The relative positions of the P1, P2, and P3 genomes in the EV71 virus genome (Genbank accession number is FJ713137) are 745-3330bp, 3331-5064bp, and 5065-7323bp, respectively.

[0212] The siRNA sequence design for P1 genome (VP4, VP2, VP3 and VP1), P2 genome (2A, 2B and 2C), P3 genome (3A, 3B, 3C, and 3D) is carried out according to the following principles:

[0213] Start at 50-100 bases behind the gene promoter of the EV71 virus FJ713137 strain, and look for a 19bp nucleotide...

Embodiment 1

[0241] Select the whole genome sequence of EV71 isolated in various regions of my country in recent years for gene comparison, obtain the EV71 virus genome (Genbank accession number is FJ713137) with a relatively high conservative sequence as a template, and then target the conserved region of the EV71 virus FJ713137 gene respectively, select 19bp nucleotide sequence, design siRNA.

[0242] The siRNA sequence design for P1 genome (VP4, VP2, VP3 and VP1), P2 genome (2A, 2B and 2C), P3 genome (3A, VPg, 3C, and 3D) is carried out according to the following principles:

[0243] Start at 50-100 bases behind the gene promoter of the EV71 virus FJ713137 strain, and look for a 19bp nucleotide sequence that meets the following conditions in the gene sequence: (1) start with G or C and end with A or T; (2) ) At least 5 of the last 7 bases at the end are A or T; (3) Avoid 4 consecutive bases, such as AAAA or CCCC, thereby increasing the complexity of bases; (4) GC content is 30-50% betwe...

Embodiment 2

[0252] Detection of inhibitory effects of siRNA sequences

[0253] (1) Culture of HEK 293a cells

[0254] Using DMEM complete medium containing 10% fetal bovine serum and 100 U / ml penicillin, 100 μg / ml streptomycin, in 24-well cell culture plate at 1 × 10 5 Inoculate HEK293a cells (from Peking University) at a density of 1 cell / well at a temperature of 37 and CO 2 The culture medium was cultured in an incubator with a content of 5% by volume, subcultured and replaced with fresh medium every 72 hours. 24 hours before transfection, cells were digested with 0.25% trypsin, counted, and then 1 × 10 5 The concentration of cells / ml was inoculated into 24-well plates, 500 μl per well.

[0255] (2) Preparation of fusion plasmid

[0256] A fusion plasmid of siRNA attack site and firefly luciferase (Firefly luciferase) expression sequence was prepared to detect the inhibition degree of siRNA to its attack site.

[0257] The specific operation steps are as follows: convert the siRNA ...

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Abstract

The invention provides siRNA (small interfering ribonucleic acid) which is capable of inhibiting expression of an enterovirus 71 type gene. The invention also provides a pharmaceutical composition which contains the siRNA provided by the invention as an active component. The invention further provides application of the siRNA in the aspect of preparing a medicament for inhibiting the expression of the enterovirus 71 type gene and application of the siRNA in the aspect of preparing a medicament for treating and / or preventing diseases. The siRNA provided by the invention has high inhibition rate on the expression of the enterovirus 71 type gene and is capable of effectively preventing and / or treating various diseases including hand-foot-and-mouth diseases.

Description

technical field [0001] The present invention relates to a siRNA for inhibiting enterovirus type 71 gene expression and a pharmaceutical composition containing the siRNA, and also relates to the application of the siRNA in the preparation of a drug for inhibiting enterovirus type 71 gene expression and its use in Application in the preparation of medicaments for the treatment and / or prevention of diseases. Background technique [0002] Enterovirus 71 (EV71) is a member of the genus Enterovirus in the Picornaviridae family and is one of the main pathogens of hand-foot-and-mouth disease (HFMD). one. In addition to causing hand, foot and mouth disease in patients, EV71 infection can also lead to various diseases related to the nervous system, such as herpetic angina, aseptic meningitis, brainstem encephalitis, and polio-like paralytic diseases. Disabling and fatality rates are high. [0003] Hand, foot and mouth disease caused by EV71 virus is a common infectious disease in i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113A61K48/00A61P31/14A61P25/00A61P11/00A61P9/00
Inventor 梁子才张鸿雁
Owner SUZHOU RIBO LIFE SCIENCE CO LTD
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