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Fluorescent cell model for screening M2 ion channel blocker for influenza virus and application method thereof

A cell model and ion channel technology, applied in the field of genetic engineering, can solve problems such as difficult high-throughput screening, limited ion channel research methods, and many influencing factors, achieving high-throughput accurate screening, clear drug action mechanism, and simple methods Effect

Inactive Publication Date: 2009-09-02
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

These experimental methods are difficult to perform high-throughput screening, and the target is unknown, the cost of animal experiments is high, and there are many influencing factors
[0004] As an ion channel, the establishment of a high-throughput screening method for M2 is still difficult: first, it is difficult to establish a model of the M2 ion channel because the M2 ion channel is toxic to cells when expressed on cells; and the existing ion channel research methods Limited, such as electrophysiology (patch clamp), ligand binding, ion flux measurement, etc., and it is still difficult to use these methods to achieve high throughput

Method used

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  • Fluorescent cell model for screening M2 ion channel blocker for influenza virus and application method thereof
  • Fluorescent cell model for screening M2 ion channel blocker for influenza virus and application method thereof
  • Fluorescent cell model for screening M2 ion channel blocker for influenza virus and application method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Example 1: Establishment of co-expression cells of H5N1 influenza A virus M2 ion channel and enhanced green fluorescent protein (EGFP)

[0031] 1. Acquisition of M2 ion channel protein gene of H5N1 influenza A virus

[0032] Using the consensus amino acid sequence of the H5N1 virus strain as a template, after optimization, the gene sequence suitable for mammalian expression was artificially synthesized (Guangzhou Funeng Gene Co., Ltd.), and named H5M2. At the same time, an expression plasmid of a drug-resistant mutant (S31N-L26I double mutation) of H5N1 influenza virus M2 was constructed, and the plasmid was named H5M2 (S31N-L26I). Both were digested with BamHI and XbaI and cloned into pcDNA4 / TO plasmid, which can only express the target gene under the induction of tetracycline in T-REx293 cells. The wild-type H5M2 gene sequence is:

[0033] CGG GAT CCAT gtc cct gct gac aga ggt gga gac ccc cac cag gaa tga gtg gga gtg cag

[0034] gtg ctc tga ctc ctc tga CCC CCT GGT G...

Embodiment 2

[0140] Example 2. Fluorescent indicator labeled M2 expressing cell model

[0141] 1. Take 2×10 cells of M2T-REx293 stable strain in good condition 6 One was inserted into a 10cm culture dish (wild type and mutant type), induced by tetracycline 2 μg / ml and cultured for 24h; in addition, the cells of the same stable strain were put into two 10cm culture dishes (wild type and mutant type), without induction, as a control . Add 10ml of complete medium and place in a carbon dioxide incubator at 37°C and 5% CO2 for constant temperature cultivation.

[0142] 2. After 24 hours, the cells were collected without trypsinization, and the supernatant was removed. The cells were gently blown down with 1 ml of PBS (0.01M, pH 7.4) buffer, and collected into a 1.5 ml EP tube. Cells were washed three times with PBS pH 7.4.

[0143] 3. The cells were resuspended with 1 ml of Hanks buffer, and labeled with biscarboxyethylcarboxyfluorescein tetraacetoxymethyl ester (BCECF-AM, final concentratio...

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Abstract

The invention provides a fluorescent cell model for screening an M2 ion channel blocker for influenza virus, and the cell model is a cell containing pH sensitive fluorescent materials. Meanwhile, the invention also provides a method for screening the M2 ion channel blocker for the influenza virus by the cell model, which is to detect the change of fluorescent values in the cell model under the condition of an acidic buffer. The invention has the advantages that the H+ flowing condition through the M2 ion channel is directly reflected by detecting the change of the fluorescent values reflecting pH in the cell; and the M2 ion channel blocker screening method established by the cell model has definite mechanism of drug action, is simple and convenient, quick and direct, and is suitable for high-flux accurate screening of the M2 ion channel blocker.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to the screening of anti-influenza drugs, more specifically to a fluorescent cell model of influenza virus M2 ion channel and a method for screening M2 ion channel blockers using the cell model. Background technique [0002] Influenza is a kind of acute viral respiratory infectious disease that is caused by influenza virus and seriously endangers human health. M2 ion channel protein is distributed on the surface of influenza virus and is a transmembrane protein composed of 97 amino acids. Four M2 ion channel protein monomers form a tetramer through disulfide bonds and hydrophobic interactions, and form an ion channel. It is an anti-influenza drug important target. Blocking the function of the M2 ion channel can effectively inhibit virus infection of cells and replication, and block the spread of virus infection. Amantadine and rimantadine, the M2 ion channel blockers currently u...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/44G01N33/15G01N21/64C12Q1/02
Inventor 陈凌李楚芳徐伟肖益平杨令芝
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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