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Application of MEK1 protein blocking agent in preparing antiviral medicine

A technology of antiviral drugs and blockers, applied in antiviral agents, pharmaceutical formulations, medical preparations containing active ingredients, etc., can solve the problems of narrow antiviral spectrum, large toxic and side effects, and easy mutation of antiviral drugs. To achieve the effect of solving the failure of vaccines and drugs

Inactive Publication Date: 2009-07-08
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Based on the research results of the present invention, those skilled in the art know based on common sense that the MEK1 protein blocker that specifically inhibits cellular MEK1 protein can inhibit virus replication that relies on the RAF / MEK / ERK signaling cascade pathway, and can overcome existing antiviral drugs. Easy to mutate, narrow antiviral spectrum, high toxicity and side effects

Method used

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  • Application of MEK1 protein blocking agent in preparing antiviral medicine
  • Application of MEK1 protein blocking agent in preparing antiviral medicine
  • Application of MEK1 protein blocking agent in preparing antiviral medicine

Examples

Experimental program
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Embodiment 1

[0051] The following descriptions are only preferred embodiments of the present invention, and are not intended to limit the protection scope of the present invention. Embodiment one: the establishment of HSV-2 infection HEK 293 cell model

[0052] 1. Reagents and consumables

[0053] Cell culture medium: Prepare high-sugar Dulbecco’s Modified Eagle Medium (DMEM) sterile culture medium according to the product instructions, and store at 4°C after aliquoting. The cell culture solution with a serum concentration of 10% and the cell maintenance solution with a serum concentration of 2% were respectively prepared for use.

[0054] Trypsin solution: use PBS to prepare trypsin solution (0.25% trypsin, 0.02% EDTA), filter and sterilize, store in 4°C after aseptically dispensing.

[0055] Culture medium: T25 plastic culture bottle, 6-well plate, 12-well plate, 96-well cell culture plate.

[0056] 2. Main instruments

[0057] American NAPCO 5% CO2 incubator, Japanese Olympus invert...

Embodiment 2

[0072] Example 2: Design and preparation of MEK1, MEK2 specific double-stranded siRNA

[0073] Using the gene sequences published in GenBank, analyze and compare the MEK1 and MEK2 gene sequences of different species by BLAST tool, select a unique conserved sequence in human genes, design and synthesize two kinds of mRNAs for MEK1 and MEK2 in human cells respectively Specific siRNAs, named siMEK1 and siMEK2 (Table 1), were used to study the effect of inhibiting mek1 or / and mek2 gene expression on HSV-2 replication. In addition, on the basis of siMEK1 and siMEK2, two bases were changed at the respective 5' ends, and two mutant siRNAs, siMut1 and siMut2, were designed as non-specific controls in transfection experiments.

[0074] The nucleotide sequence positions of siMEK1 and siMut1 were determined according to the mek1 gene sequence in GenBank (GenBank accession number NM_002755.2). The positions of siMEK2 and siMut2 nucleotide sequences were determined according to the mek2 g...

Embodiment 3

[0087] Example 3: Establishment of a cell model for knockdown (knock-down) mek1 mRNA and / or mek2 mRNA targets

[0088] (1) Transient transfection experiment:

[0089] The day before transfection, HEK293 cells were seeded in 35mm culture dishes at a seeding density of 1×10 5 Each well, add 2ml of culture medium to each well. After 24 hours, when the cells have grown to cover 30-50% of the bottom area of ​​the culture dish, they are ready for transfection. Prepare the following solution in a 1.5ml centrifuge tube: 20-30nM siRNA is dissolved in 200μl serum-free and double-antibody-free DMEM culture medium, and gently mix for 10 seconds. Add 8-12 μl of transfection reagent (INTERFERin transfection reagent) to 200 μl of serum-free and double-antibody-free DMEM culture medium. Let stand at room temperature for 10 minutes. After washing the cells with DMEM medium without serum and double antibody, add 1 ml of DMEM medium containing 10% FBS preheated at 37°C. The above mixture wa...

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Abstract

The invention relates to an MEK1 protein blocker and application thereof in preparing antiviral drugs. The MEK1 protein blocker provided by the invention can lower MEK1 protein level in cells through specificity or inhibit activation of MEK1 protein through specificity, so as to inhibit viral multiplication relying on host cell RAF / MEK / ERK signal channels. The invention specifically provides an MEK1 protein blocker which is a siRNA molecule combined with specificity of mek1 mRNA. The SODN nucleotide sequence thereof is shown as SEQ ID No: 1, and the ASODN nucleotide sequence thereof is shown as SEQ ID No: 2. The MEK1 protein blocker can inhibit viral multiplication relying on host cell RAF / MEK / ERK signal channels without affecting MEK2 protein level and functions, has single anti-virus target, security to organisms and wide antivirus spectrum, can overcome the problem of virus variation, and can be developed into a novel anti-virus drug.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the application of a protein blocker in the preparation of antiviral drugs, in particular to the application of MEK1 protein blocker in the preparation of antiviral drugs. Background technique [0002] There are two main types of antiviral drugs currently in use. One category is antiviral drugs designed for the components of the virus itself. Most of the currently used antiviral drugs belong to this category, such as lamivudine, which is a nucleoside analog that can inhibit the DNA synthesis of viruses, and its antiviral effect clear. However, such drugs target the nucleic acid components of the virus, which can easily cause the virus to mutate and develop drug resistance. The other is antiviral drugs derived from the host's natural antiviral substances. At present, interferon is mainly used clinically, and it is mainly used in combination with other antiviral drugs, and it is only ...

Claims

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Application Information

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IPC IPC(8): A61K31/7088A61P31/22
Inventor 彭宜红张浩罗志军
Owner PEKING UNIV
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