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Cell models and screening methods for screening calcium-activated chloride channel inhibitors

A chloride ion channel and screening method technology is applied to the cell model for screening calcium-activated chloride ion channel inhibitors and the field of high-throughput screening. , cost-effective, sensitive and specific screening effects

Inactive Publication Date: 2016-08-17
JINLIN MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Although the patch clamp technique is the gold standard for discovering Anoctamin1 and Anocatmin2 calcium-activated ion channel inhibitors, the patch clamp technique not only requires relatively expensive special equipment and related technical personnel, but also has low efficiency and is not suitable for Anoctamin1 and Anoctamin2. High-throughput screening of Anocatmin2 calcium-activated chloride channel inhibitors

Method used

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  • Cell models and screening methods for screening calcium-activated chloride channel inhibitors
  • Cell models and screening methods for screening calcium-activated chloride channel inhibitors
  • Cell models and screening methods for screening calcium-activated chloride channel inhibitors

Examples

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Embodiment 1

[0040] The high-throughput screening method for Anoctamin1 inhibitors of the present invention comprises the following steps in turn:

[0041] (1) Anoctamin1 and YFP-H148Q / I152L eukaryotic expression vectors were constructed. The resistance of the constructed Anoctamin1 eukaryotic expression vector was Blasticidin, while the resistance of YFP-H148Q / I152L eukaryotic expression vector was Hygromycin.

[0042] Taking Anoctamin1 as an example, such as figure 1 , figure 2 As shown, the results of the double enzyme digestion map and the sequencing map confirmed that Anoctamin1 was successfully cloned into the eukaryotic expression vector pUB6 / V5.

[0043] The eukaryotic expression vector of YFP-H148Q / I152L is pcDNA3.1-YFP-H148Q / I152L.

[0044] (2) The above two vectors were stably transfected into FRT cells suitable for high-throughput screening, so that the FRT cells co-expressed Anoctamin1 and YFP-H148Q / I152L proteins. And after 3 limited dilutions, FRT cell clones with high p...

Embodiment 2

[0057] Anoctamin1 in Example 1 was replaced with Anoctamin2, and other conditions were exactly the same as in Example 1, and the screening test was done, and the results obtained were exactly the same.

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Abstract

The invention provides a cell model and a screening method for screening a calcium-activated chloride ion channel inhibitor. The screening method sequentially comprises the steps of (1) constructing eukaryotic expression vectors of Anoctamin1 or Anocatmin2 and YFP-H148Q / I152L; (2) stably transfecting the two vectors into FRT (Fisher rat thyroid) cells respectively to ensure that the RFT cells co-express two proteins of YFP-H148Q / I152l and Anoctamin1 or Anocatmin2, and performing multiple times of limiting dilution to obtain an FRT cell model; (3) transferring the FRT cell model to a black-wall microporous plate with a black wall and a transparent bottom; (4) adding a small molecular compound to be detected into the black-wall microporous plate, incubating, then adding a regent for raising the concentration of calcium ions in cells and high-concentration iodide ions, and screening the calcium-activated chloride channel inhibitor by detecting the change of relative fluorescence intensity.

Description

technical field [0001] The invention belongs to the fields of biology and pharmacy, and specifically relates to a cell model for screening calcium-activated chloride channel inhibitors and a high-throughput screening method thereof. Background technique [0002] As early as the 1980s, scientists recorded the classic calcium-activated chloride channel current on Xenopus laevis oocytes, confirming the existence of calcium-activated chloride channels (Calcium-activatedChloride Channels, CaCCs). At present, it is known that CaCCs widely exist in various excitatory and non-excitatory cells, play an important role in various physiological functions and pathological processes, and are potential candidates for diseases such as hypertension, cystic fibrosis, diarrhea and some specific tumors. drug targets. The molecular identity of the calcium-activated chloride ion channel was discovered by three independent laboratories in 2008 --- transmembrane protein 16A (TMEM16A) is a true cal...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/85C12Q1/02
Inventor 郝峰扈昕虹方芳杨敬研郑锴曲适杨娟王长文孙宝霄李艳许会静孙美艳陈爽于向博王彦力
Owner JINLIN MEDICAL COLLEGE
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