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Establishing method for drug screening model of high-throughput Japanese encephalitis virus helicase inhibitor

A Japanese encephalitis virus and helicase inhibitor technology, applied in the field of biomedicine, can solve the problem that the Japanese encephalitis virus helicase drug design model has not been established, and no Japanese encephalitis virus helicase drug report has been reported. and other problems, to achieve the effect of stable reaction, simple and easy method, and easy completion.

Inactive Publication Date: 2013-03-27
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, no drug design model for JEV helicase has been established, and there are no reports of drugs targeting JEV helicase

Method used

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  • Establishing method for drug screening model of high-throughput Japanese encephalitis virus helicase inhibitor
  • Establishing method for drug screening model of high-throughput Japanese encephalitis virus helicase inhibitor
  • Establishing method for drug screening model of high-throughput Japanese encephalitis virus helicase inhibitor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Optimization of detection conditions for a drug screening model of a high-throughput Japanese encephalitis virus helicase inhibitor

[0047] (1) Preparation of helicase protein

[0048] The NS3 gene of Japanese encephalitis virus (Genbank accession number: U47032) was artificially synthesized, and the helicase fragment was digested with Nco I and Xho I, respectively, and the E. coli prokaryotic expression vector pET30a, T4 DNA Ligase (TakaRa, Dalian Bao Biological Engineering Co., Ltd. ) at 16°C overnight to obtain the recombinant plasmid pET30a-helicase, and the constructed recombinant plasmid pET30a-helicase was transformed into Escherichia coli BL21 (DE3) PlysS for expression. Collect the expressed protein supernatant with Ni 2+ Affinity purification, the purified helicase protein was identified by SDS-PAGE and Western blot, then glycerol was added to a final concentration of 20% (v / v), the concentration was determined by BCA method, and frozen at -80°C f...

Embodiment 2

[0069] Embodiment 2: the application of a drug screening model of a high-throughput Japanese encephalitis virus helicase inhibitor, its steps are as follows:

[0070] (1) Add 3uM of the helicase protein prepared in Example 1, the compound to be screened and the reaction buffer (buffer for dissolving protein, 20mM Tris-base, 150mM Tris-base, 150mM Nacl, 5% glycerol), mix and incubate for 5 minutes, add F chain 200nM and Q chain 240nM, incubate for another 5 minutes, add C chain 2.4uM and ATP 2mM, react at 37°C for 90 minutes, add reaction stop solution, in The fluorescence intensity in the system is detected in the fluorescence detector. At the same time, set up a control group without adding the compound to be screened (group C) and a control group without adding the substrate (group C1).

[0071] (2) Calculate the inhibition rate of the compound: inhibition rate = 1-(average fluorescence of experimental group-average fluorescence of group C1)÷(average fluorescence of group C...

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Abstract

The invention discloses an establishing method for a drug screening model of high-throughput Japanese encephalitis virus helicase inhibitor. The establishing method comprises the following steps: 1, expression and purification of helicase protein: artificially synthesizing Japanese encephalitis virus NS3 gene, using NcoI and XhoI to respectively digest helicase segments; connecting the digested helicase segments with escherichia coli prokaryotic expression vector over the night to obtain a helicase protein with a sequence shown as SEQ ID No. 1; collecting liquid supernatant of the expression protein, using Ni2+ affinity chromatography to purify the helicase protein, using a BCA process to measure the protein concentration; 2, designing helicase DNA substrate; 3, adding helicase protein 3uM, compound to be sieved and reaction buffer solution to black 96 holes for mixing and incubating, detecting fluorescence intensity in the detecting system by a fluorescence detector, not adding a control group of the substrate; and 4, calculating the inhibition ratio of the compound. The establishing method is simple and easy to carry out, has high flexibility, low cost, and less samples, can monitor the reaction process of helicase, and has a great meaning in developing the Japanese encephalitis virus medicine in the future.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a method for establishing a drug screening model of a high-throughput Japanese encephalitis virus helicase inhibitor, which can be used for drug screening. Background technique [0002] Japanese encephalitis virus not only causes central nervous system diseases in humans, but also causes reproductive disorders in pigs. It is an important pathogen of zoonotic infectious diseases. Japanese encephalitis virus mainly invades the nervous system, including the thalamus, basal ganglia, brainstem, cerebellum, cerebral cortex, and spinal cord, resulting in severe central nervous system diseases such as polio-like severe paralysis, aseptic meningitis, and cerebral palsy. Inflammation, 71% of patients are basal ganglia and thalamus damage, severe movement disorders, 43% of patients with brainstem damage, often acute respiratory failure symptoms, or even death. The fatality rate and cli...

Claims

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Application Information

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IPC IPC(8): C12Q1/25C12N15/52C12N15/70C12N9/00G01N21/64
Inventor 宋云峰方金娥曹胜波陈焕春
Owner HUAZHONG AGRI UNIV
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