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Structure and applications of oligonucleotide of EV71, Dengue, Japanese encephalitis and flu virus of target CDK1 resistance

An oligonucleotide and dengue virus technology, applied in antiviral agents, recombinant DNA technology, DNA/RNA fragments, etc., can solve the problem of no anti-EV71 virus, etc., achieve important social and economic benefits, and have small toxic and side effects , highly specific effect

Inactive Publication Date: 2015-04-15
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Studies have confirmed that cyclin-dependent kinase 1 (CDK1) is one of the important targets of anti-tumor drugs. At present, it has been reported that CDK1 inhibitors have anti-herpes zoster virus, cytomegalovirus, and HIV effects. However, there are no reports of anti-EV71 virus, dengue virus, Japanese encephalitis virus and influenza virus infection

Method used

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  • Structure and applications of oligonucleotide of EV71, Dengue, Japanese encephalitis and flu virus of target CDK1 resistance
  • Structure and applications of oligonucleotide of EV71, Dengue, Japanese encephalitis and flu virus of target CDK1 resistance
  • Structure and applications of oligonucleotide of EV71, Dengue, Japanese encephalitis and flu virus of target CDK1 resistance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] This example mainly illustrates the design, synthesis and screening of antisense oligonucleotides targeting human CDK1 gene against EV71 virus.

[0043] Materials and Methods

[0044] 1. Design and synthesis of antisense oligonucleotides CDK1-1-5

[0045] Combined with the human gene expression profile of anti-EV71 (expression profile number GSE16358) and HPRD (human protein interaction network), a core subnetwork containing more than 100 nodes was designed by using the method of subnetwork identification, and then topologically important The node was screened out for the gene CDK1. All oligonucleotides were synthesized with 8909 type automatic DNA synthesizer to synthesize antisense oligonucleotides modified with full sulfur. After the synthesis was completed, the concentrated ammonia solution was cleaved at 55°C and deprotected for 15 hours, then purified by a Micro Pure II reverse-phase purification column (Oligo Prep OP120, SAVANT), quantified by ultraviolet light...

Embodiment 2

[0064] This example mainly illustrates the specificity of anti-EV71 virus of CDK1-4 after designing the sense of CDK1-4 and random verification.

[0065] Materials and methods

[0066] 1. Design and synthesis of sense strand and random strand of CDK1-4

[0067] The positive-sense strand is directly obtained by base complementation with the antisense oligonucleotide antisense sequence of CDK1-4, and its random strand is designed by using SMS2 (The Sequence Manipulation Suite 2), a design software such as DNA random protein, and Through online blast sequence alignment with GeneBank, it will not interfere with the expression of other normal human genes.

[0068] 2. Specific verification of CDK1-4 anti-EV71 virus

[0069]Referring to Example 1, the RD cells were plated on a 96-well cell culture plate, and 75-80% of them were confluent the next day. Replace 75-80% RD cells with maintenance solution containing 2% fetal bovine serum for use. Set up five concentration gradients of...

Embodiment 3

[0075] This example mainly illustrates that CDK1-4 can dose-dependently inhibit the RNA of EV71 virus

[0076] Materials and methods

[0077] CDK1-4 inhibits the dose-dependent effect of EV71 viral RNA

[0078] The RD cells were plated on a 96-well cell culture plate, and 75-80% of them were confluent the next day. The RD cells that were 75-80% full were replaced with maintenance solution containing 2% fetal bovine serum, and CDK1-4 of different concentrations were added. Four concentration gradients of 0.25, 0.5, 1.0, and 2.0 μM were set up for CDK1-4, respectively, and three secondary wells were set up for each sequence concentration, and a positive control group for virus and a negative control group for RD cells were set up. After acting for 60 minutes, add 3.3 μl of EV71 virus dilution solution of 100 TCID50 / 0.1 ml to each well, and incubate at 37° C. for 2 days. After 2 days, the supernatant of the culture medium was collected, and viral RNA was extracted using a vira...

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Abstract

The invention relates to a structure and applications of antisense oligonucleotide, in particular to targeting cyclin-dependent kinase 1, a structure of antisense oligonucleotide of Enterovirus 71, Dengue virus and Japanese encephalitis resistance and applications of the antisense oligonucleotide in preparing medicines for treating Enterovirus 71, Dengue virus, Japanese encephalitis, flu virus and related diseases.

Description

Technical field: [0001] The invention relates to the field of bioengineering drugs, in particular to a treatment targeting cyclin-dependent kinase 1 (CDK1, Cyclin-dependent kinases 1) for EV71 (Human enterovirus 71), dengue The sequence, structure and therapeutic drug of antisense oligonucleotide (ASODN, antisense oligodexynucleotide) infected by virus (Dengue virus), encephalitis virus or influenza virus. Background technique [0002] EV71 virus, dengue virus, encephalitis virus, and influenza virus all belong to RNA viruses, and their infection hazards are very serious. [0003] EV 71 virus is highly infective and has a high morbidity rate, especially neurological complications. The disease is easy to spread, prone to epidemics, and difficult to prevent and control. In the mid-1970s, Bulgaria, Hungary and other countries successively broke out the epidemic of EV71 hand, foot and mouth disease with central nervous system disease as the main clinical feature, resulting in ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113A61K48/00A61P31/14A61P31/16
Inventor 王升启杨静伯晓晨刘娟张莉李康何丽娜
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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