Structure and application of target RAF1 (c-Raf, v-raf-leukemia viral oncogene 1) anti-encephalitis-b-virus oligonucleotides

An oligonucleotide, Japanese encephalitis virus technology, applied in antiviral agents, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of high price and large side effects, and achieve important social and economic benefits.

Inactive Publication Date: 2012-11-14
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

However, currently there is no specific treatment method and effective drug for JE clinically, and vaccination is still the only and most important control measure.
The inactivated vaccines currently used for immune prevention mainly include purified mouse brain inactivated vaccines and hamster kidney cell inactivated vaccines. They have been widely used for many years and have played a very important role in reducing the incidence of Japanese encephalitis. However, the side effects are large. higher price
Live vaccines and live attenuated vaccines have more advantages than inactivated vaccines, but the safety and quality control of the production process of this vaccine has not been recognized internationally, so more information needs to be accumulated on its safety and other aspects

Method used

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  • Structure and application of target RAF1 (c-Raf, v-raf-leukemia viral oncogene 1) anti-encephalitis-b-virus oligonucleotides
  • Structure and application of target RAF1 (c-Raf, v-raf-leukemia viral oncogene 1) anti-encephalitis-b-virus oligonucleotides
  • Structure and application of target RAF1 (c-Raf, v-raf-leukemia viral oncogene 1) anti-encephalitis-b-virus oligonucleotides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] This example mainly illustrates the design, synthesis and screening of antisense oligonucleotides targeting human RAF1 gene against Japanese encephalitis virus.

[0033] Materials and Methods

[0034] 1. Design and synthesis of antisense oligonucleotides targeting RAF1 gene For RAF1 mRNA (NM_002880), 20 antisense oligonucleotides targeting RAF1 were designed using the online server Soligo, and all sulfur was synthesized by a 3900 automatic DNA synthesizer Modified antisense oligonucleotides. After the synthesis was completed, the concentrated ammonia solution was cleaved and deprotected at 55°C for 15 hours, then purified by a Micro PureII reverse-phase purification column (Oligo Prep OP120, SAVANT), quantified by ultraviolet light, dried in vacuum, and stored at -20°C for future use.

[0035] 2. BHK21 cells, virus and control drug The virus was amplified by infecting BHK21 cells, then mixed, aliquoted and stored at -70°C for later use. The JE virus strain used was Ji...

Embodiment 2

[0045] This example mainly illustrates the specificity and dose-dependence of RAF1-19 in resisting CPE caused by JE virus and inhibiting the replication of JE virus at the cellular level.

[0046] Materials and methods

[0047] 1. The toxicity detection of ODNs refers to Example 1. BHK21 cells are plated on a 96-well cell culture plate, and 80%-90% of the BHK21 cells are replaced with a maintenance solution containing 0.7% fetal bovine serum, and different concentrations of antisense are added. Six concentration gradients of 0.5, 1, 2, 4, 8, and 16 μM were set up for RAF1-19 and RAF1-19 respectively, and three replicate wells were set up for each concentration, and a negative control group of BHK21 cells was set up, and cultured at 37°C for 3 days. Cell Counting Kit-8 (DoJinDo product) was used to measure the degree of cytopathic disease (expressed as OD450) on a microplate reader (BIO-RAD product, model: Model 680), and the influence of RAF1-19 on the cells was observed.

[...

Embodiment 3

[0059] This example mainly illustrates the evaluation of anti-JEV activity in RAF1-19 mice.

[0060] Materials and Methods

[0061] A number of SPF three-week-old BALB / C female mice were ordered, and the mice were weighed and grouped in advance at the beginning of the experiment, with eight mice in each group, generally divided into PBS control group, JEV virus infection group and antisense drug group ( Specifically grouped according to the dose concentration), and prepare the things used in the experiment: 1mL syringe, cotton swab, electronic scale, record book. During the experiment, both the PBS control group and the JEV virus infection group were injected with a certain amount of liquid according to body weight in one side of the abdominal cavity for five consecutive days, and the antisense drug group was injected with a certain amount of virus on the first day, followed by an appropriate amount of drugs. , followed by injection of the same dose of drugs for the next four...

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Abstract

The invention relates to a structure and application of antisense oligodexynucleotide (ASODN) and particularly relates to a structure of the ASODN discovered with a target RAF1 (c-Raf, v-raf-leukemia viral oncogene 1) and application of the ASODN in preparation of drugs for treating encephalitis b virus infections and related diseases.

Description

technical field [0001] The invention relates to the field of bioengineering drugs, in particular to a method targeting RAF1 protein kinase (RAF1, c-Raf, v-raf-leukemia viral oncogene 1) for the treatment of epidemic Japanese encephalitis virus (referred to as Japanese encephalitis virus, The sequence, structure and therapeutic drug of antisense oligonucleotide (ASODN, antisense oligodexynucleotide) infected by JEV). Background technique [0002] JEV belongs to the Flavivirus genus in the family Flaviviridae. Epidemic Japanese encephalitis (referred to as JE) is internationally known as Japanese encephalitis (Japanese encephalitis). The disease is an acute infectious disease of the central nervous system caused by the Japanese encephalitis virus (Japanese encephalitis virus) through mosquito bites, and is also a zoonotic natural foci disease. Clinically, it is characterized by high fever, disturbance of consciousness, convulsions, respiratory failure and meningeal irritatio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K48/00A61P31/14
Inventor 王升启杨静张莉伯晓晨何丽娜韩明明李康
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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