Rapid fluorescence screening method for marine neurotoxin based on sodium ion channel Nav1.1

A neurotoxin and screening technology, applied in fluorescence/phosphorescence, material excitation analysis, etc.

Pending Publication Date: 2021-11-05
CHINA NAT CENT FOR FOOD SAFETY RISK ASSESSMENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] At present, there is no report of a rapid fluorescence detection method based

Method used

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  • Rapid fluorescence screening method for marine neurotoxin based on sodium ion channel Nav1.1
  • Rapid fluorescence screening method for marine neurotoxin based on sodium ion channel Nav1.1
  • Rapid fluorescence screening method for marine neurotoxin based on sodium ion channel Nav1.1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Embodiment 1Nav1.1-CHO cell culture and experimental preparation

[0066] In this experiment, a CHO cell line stably expressing Nav1.1 channel was selected, that is, Nav1.1-CHO (Nav1.1 gene information: human NaV1.1 (SCN1A), NM_001165963, CDS size is 6030bp). In a cell culture dish, use F-12 medium containing 10% FBS, 1% penicillin / streptomycin mixed solution (100×) and 200 μg / ml hygromycin B at a constant temperature of 37°C and 5% CO 2 cultured in a cell culture incubator. Transfer Nav1.1-CHO stably transfected cells to 96-well cell culture plate, 5×10 per well 4 cells, 96-well plates were coated overnight with PLL in advance. After plating for about 18 hours, the cells grow to about 80% and can be detected.

Embodiment 2

[0067] Example 2 Rapid preliminary screening of marine neurotoxins——primary screening of agonists

[0068] ① Dilute the membrane potential red fluorescent dye with HHBS buffer at a ratio of 1:10 to prepare a fluorescent dye working solution;

[0069] ②Dilute each stock solution of the compound to be tested with HHBS buffer solution to a suitable single-point concentration (5×final concentration, recommended 500nM~10μM);

[0070] ③ Take the 96-well plate containing the Nav1.1-CHO cells to be tested out of the incubator and place at room temperature;

[0071] ④Remove the culture medium of the original well, add 100 μl HHBS buffer solution to each well, add 100 μl fluorescent dye working solution to each well, incubate in a 37°C incubator for 30 minutes, and then place at room temperature for 30 minutes;

[0072] ⑤ Microplate reader detection, the experimental parameters of the microplate reader are set as follows: excitation light: 620nm / 10; emission light: 665nm / 8. 50 μl / well...

Embodiment 3

[0073] Example 3 Rapid Preliminary Screening of Marine Neurotoxins—Preliminary Screening of Inhibitors

[0074] ① Dilute the membrane potential red fluorescent dye with HHBS buffer at a ratio of 1:10 to prepare a fluorescent dye working solution;

[0075] ②Dilute the veratridine stock solution with HHBS buffer solution into a working solution (5×final concentration), namely 150 μM;

[0076] ③Dilute the stock solution of each compound to be tested with HHBS buffer to an appropriate single-point concentration (12.5×final concentration, recommended 1μM-20μM);

[0077] ④ Take the 96-well plate containing the Nav1.1-CHO cells to be tested out of the incubator and place at room temperature;

[0078] ⑤Remove the medium of the original well, add 80 μl HHBS buffer solution to each well, add 100 μl fluorescent dye working solution to each well, incubate at 37°C for 30 minutes, and then place at room temperature for 30 minutes;

[0079] ⑥ Microplate reader detection, the experimental p...

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Abstract

The invention provides a rapid fluorescence screening method for marine neurotoxin. The rapid screening method comprises the following steps of (1) screening an agonistic effect, (2) screening the inhibiting effect, and (3) generating a report. The sodion channel Nav1.1 targeting toxicity determination fluorescence screening technology is established, the primary screening of the agonist and the inhibitor can be rapidly performed, the agonist/inhibition curve and the EC50/IC50 value can be obtained, and the strategy is provided for the low-cost rapid screening of the marine neurotoxin.

Description

technical field [0001] The present disclosure relates to the field of biotechnology, in particular to a method for rapid screening of marine neurotoxin fluorescence based on sodium ion channel Nav1.1. Background technique [0002] Marine toxins are special metabolic components in marine organisms, most of which are highly toxic, mainly produced by phytoplankton or microorganisms, and can accumulate and enrich in specific species through the food chain. Seafood is an important food source for humans, and marine toxin poisoning incidents caused by eating seafood are not uncommon. Among the many marine toxins, marine neurotoxins are the most toxic and lethal, mainly including: tetrodotoxin, saxitoxin, brevetoxin, sija toxin, sea anemone toxin, etc., and their similar structures thing. Most of them specifically act on voltage-gated sodium channels (Voltage-gated sodium channels, VGSCs) on nerve and muscle cell membranes, thereby affecting the conduction of action potentials an...

Claims

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Application Information

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IPC IPC(8): G01N21/64
CPCG01N21/6428G01N2021/6432
Inventor 周爽邱楠楠张烁郑双佳李英骥
Owner CHINA NAT CENT FOR FOOD SAFETY RISK ASSESSMENT
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