阅读说明：本技术 一种基于钠离子通道Nav1.1的海洋神经毒素荧光快速筛查方法 (Rapid marine neurotoxin fluorescence screening method based on sodium ion channel Nav1.1 ) 是由 周爽 邱楠楠 张烁 郑双佳 李英骥 于 2021-08-05 设计创作，主要内容包括：本公开提供了一种海洋神经毒素荧光的快速筛查方法,包括：(1)激动作用筛查；(2)抑制作用筛查；(3)生成报告。本公开建立了钠离子通道Nav1.1靶向毒性测定荧光筛查技术,可快速进行激动剂和抑制剂初筛,获得激动/抑制曲线及EC50/IC50值,为海洋神经毒素的低成本快速筛查提供策略。(The present disclosure provides a rapid screening method for marine neurotoxin fluorescence, comprising: (1) screening for agonism; (2) screening inhibition; (3) a report is generated. The invention establishes a sodium ion channel Nav1.1 targeted toxicity determination fluorescence screening technology, can quickly perform primary screening on an agonist and an inhibitor, obtains an agonistic/inhibitory curve and an EC50/IC50 value, and provides a strategy for low-cost quick screening of marine neurotoxin.)

1. A rapid screening method for marine neurotoxin fluorescence is characterized by comprising the following steps:

(1) screening for agonism: adding a compound to be detected and a negative control and a positive control into a CHO cell containing Nav1.1, and detecting by an enzyme-labeling instrument;

(2) screening for inhibition: adding a compound to be detected, a negative control and a positive control into a CHO cell containing Nav1.1, adding a given dose of agonist veratridine, and detecting by an enzyme-labeled instrument;

(3) a report is generated.

2. The rapid screening method according to claim 1, wherein in step (1), the negative control is HHBS buffer and the positive control is veratridine.

3. The rapid screening method of claim 1, wherein the negative control is HHBS buffer and the positive control is tetrodotoxin.

4. The rapid screening method according to claim 1, wherein step (1) comprises:

preparing a fluorescent dye working solution;

diluting a stock solution of a compound to be detected containing the compound to be detected into a working solution to be detected;

③ culturing cells: culturing CHO cells containing Nav1.1;

removing liquid in the cell culture hole, adding a fluorescent dye working solution and an HHBS buffer solution into the cell culture hole, incubating in an incubator at 37 ℃ for 30min, and standing at room temperature for 30 min;

detecting by an enzyme labeling instrument: adding a working solution to be detected, a negative control and a positive control into the CHO cells containing Nav1.1 respectively, and detecting.

5. The rapid screening method according to claim 4, wherein in step (r), the membrane potential red fluorescent dye is applied in a ratio of 1: diluting with HHBS buffer solution at a ratio of 10 to prepare fluorescent dye working solution.

6. The rapid screening method according to claim 4, wherein in step (II), the stock solution of the compound to be tested is diluted to a suitable single-site concentration of 1 μ M to 20 μ M with HHBS buffer solution as the working solution to be tested.

7. The rapid screening method according to claim 4, wherein in step (c), the CHO cells used are CHO cells containing Nav1.1, the gene information of Nav1.1: human nav1.1(SCN1A), NM — 001165963, CDS size 6030 bp.

8. The rapid screening method according to claim 4, wherein the stock solution of the compound to be tested is diluted with HHBS buffer solution by 8-10 gradient concentrations step by step to serve as the working solution to be tested for standby, and during the determination, the working solution to be tested with the gradient concentrations is sequentially determined hole by hole to draw the fluorescence-concentration excitation curve.

9. The rapid screening method according to claim 1, wherein step (2) comprises:

preparing a fluorescent dye working solution;

diluting veratridine stock solution with HHBS buffer solution to obtain veratridine working solution;

diluting the compound to be detected into working solution to be detected;

fourthly, cell culture: culturing CHO cells containing Nav1.1, plating for about 18h, and detecting when the cells grow to about 80%;

fifthly, removing liquid in the cell culture hole, adding a fluorescent dye working solution and an HHBS buffer solution into the cell culture hole, incubating for 30min in an incubator at 37 ℃, and then standing for 30min at room temperature;

sixth, enzyme-linked immunosorbent assay: adding a working solution to be detected, a negative control and a positive control into the CHO cell containing Nav1.1 respectively, adding a veratridine working solution, and detecting.

10. The rapid screening method according to claim 8, wherein the stock solution of the compound to be tested is diluted with HHBS buffer solution by 8-10 gradient concentrations step by step to serve as the working solution to be tested for standby, and during the determination, the working solution to be tested with the gradient concentrations is sequentially determined hole by hole to draw the inhibition curve of fluorescence-concentration.

Technical Field

The disclosure relates to the technical field of biology, in particular to a rapid fluorescence screening method for marine neurotoxin based on a sodium ion channel Nav1.1.

Background

Marine toxins (marine toxins) are specific metabolic components of the marine organism, mostly strongly toxic, are produced mainly by phytoplankton or microorganisms, and can accumulate and concentrate in specific species through the food chain. Marine products are an important food source for human beings, and marine toxin poisoning events caused by eating marine products are rare. Among the marine toxins, the most toxic and lethal is the marine neurotoxin, which mainly comprises: tetrodotoxin, saxitoxin, gymnodin, ciguatoxin, actinocongestin, and the like, and structural analogs thereof. They mostly act on Voltage-gated sodium channels (VGSCs) on the cell membranes of nerves and muscles specifically, thereby affecting action potentials and the conduction of excitation signals between nerves and muscles, generating severe acute poisoning symptoms of nervous system, respiratory system and cardiovascular system, and even leading to death.

The research optimizes and establishes a sodium ion channel Nav1.1 targeted toxicity determination Fluorescence sensing technology based on the obtained CHO cells stably expressing the sodium ion channel Nav1.1 of the human nerve cells and the commercialized Red fluorescent dye AAT Bioquest, Quest Membrane Potential Assay Kit Red Fluorescence sensitive to transmembrane Potential, and can realize marine neurotoxin screening, action type (agonism and inhibition) identification and semi-quantitative determination of toxicity equivalent rapidly and in high flux. The technology can be used for discovery and early warning, high-throughput screening and preliminary identification of toxin action types and toxicity of unknown marine neurotoxin, and can also be used in the fields of preliminary screening of related drugs and the like.

The voltage-gated sodium ion channel is a membrane protein widely existing in tissues such as nervous system, heart, muscle and the like of animals, can excite cell membrane, and participates in the generation and conduction of action potential. To date, 9 subtypes have been identified separately in mammals: nav1.1 to Nav1.9. The protein consists of an alpha subunit and a beta subunit, wherein the alpha subunit consists of four symmetrical homologous repeating functional regions (I-IV) to form a sodium ion transmission pore channel structure. At present, six toxin action targets are found on mammalian neurons VGSCs, distributed on the outer side of cell membranes of alpha subunits, the inner side of channel holes and on transmembrane segments and sequentially named as sites 1 to 6(Site 1 to Site 6). Wherein Nav1.1 is mainly present in human nerve cells, is a target point of specific action of a plurality of nerve drugs and neurotoxins, and shows antagonist or agonist action after being combined, thereby influencing action potential and conduction of excitation signals between nerves and muscles.

Although the marine neurotoxin has different structures, the marine neurotoxin can act on VGSCs with high selectivity and high affinity and is presented as an antagonist or an agonist, thereby causing acute poisoning symptoms of a nervous system, a respiratory system and a cardiovascular system and even leading to death.

The existing methods for detecting marine neurotoxins can be mainly divided into methods for structural determination based on analytical chemistry and methods for toxicity determination based on toxicology. The structure determination method mainly depends on analysis technologies such as chromatography, mass spectrometry and the like, and the types and the content of toxins are accurately determined based on toxin standard substances. The toxicity determination method comprises the following steps: (1) the toxicity effect-based determination methods, such as mouse animal experiments, cytotoxicity analysis, and the like, reflect the toxicity of the compound according to the toxic symptoms. (2) The interaction of the compound with a toxic target (e.g., sodium channel) is measured, indicating its toxicity.

Existing methods for detecting marine neurotoxins mainly include two major classes: toxicology-based toxicity assays (e.g., mouse bioassay, cytotoxicity assays, receptor-targeted binding assays) and analytical chemistry-based structural assays (e.g., LC-MS).

The structure determination method generally adopts a chromatography-mass spectrometry combined technology to carry out qualitative and quantitative determination according to a toxin standard substance; but do not allow screening assays for toxins that lack standards, have undefined structures of the target toxin, and are completely unknown. A mouse biological experiment and a cytotoxicity analysis method based on a toxicity effect can reflect the comprehensive toxicity effect of a compound, can indicate the existence of unknown toxin, but cannot identify the action type of the toxin, and have long experiment period, particularly large consumption of the compound to be detected by an animal experiment method.

The receptor targeted binding analysis indicates the toxicity of the compound by directly or indirectly measuring the interaction between the compound and a sodium ion channel target spot, can determine the action type, has relatively short measuring time, and can realize rapid detection by designing and optimizing a proper signal sensing system.

The patch clamp technology is the main technology for researching the interaction between a compound and an ion channel at present, and the method comprises the steps of sealing a cell membrane containing the ion channel through negative pressure attraction by a glass microelectrode pipette, and measuring the ion current of the ion channel to reflect the molecular motion of single or multiple ion channels of the cell membrane. The patch clamp technique is called "gold standard" for studying ion channels and is widely used in modern cell electrophysiological studies. However, the patch clamp technology has high requirements on instruments and personnel operation, low screening flux and high cost, and restricts the application of large-scale screening. Therefore, the development of a rapid, high-throughput and low-cost screening technology taking the ion channel Nav1.1 of the nervous system as a target is of great significance to the discovery and identification of the marine neurotoxin.

Marine neurotoxins exert toxic effects by interacting with sodium ion channels, affecting the transmembrane potential and sodium current of cells in the mode of agonists or inhibitors. Thus, their interaction can be indicated in three ways: (1) direct monitoring of sodium current (e.g., patch clamp technique), (2) determination of changes in sodium ion concentration in cells, and (3) determination of changes in transmembrane potential.

Detection methods for determining changes in intracellular sodium ion concentration require a specific sodium indicator dye that emits a fluorescent signal upon binding to sodium ions, and several dyes are currently commercially available. They are all provided in the form of Acetoxymethyl (AM) groups, enabling passive diffusion across the cell membrane. Once inside the cell, the AM ester is hydrolyzed by endogenous esterases, thereby trapping the dye inside the cell. In this method, the extracellular sodium indicator dye must be washed away or some quencher added to eliminate extracellular fluorescence.

At present, a fluorescence rapid detection method which takes a Nav1.1 channel as a target point and takes monitoring of transmembrane potential as a principle is not reported. According to the scheme, on the basis of stable expression of the CHO cells of the Nav1.1 ion channel protein, red fluorescent dye sensitive to transmembrane potential is adopted, experimental conditions are optimized, a sodium ion channel Nav1.1 targeted toxicity determination fluorescence screening technology is established, primary screening of an agonist and an inhibitor can be rapidly carried out, an agonistic/inhibitory curve and an EC50/IC50 value are obtained, and a strategy is provided for low-cost rapid screening of marine neurotoxin.

Disclosure of Invention

The present disclosure adopts CHO cells (Nav1.1-CHO) stably expressing sodium ion channel Nav1.1 of human nerve cells and commercial Red fluorescent dye AAT Bioquest, Quest Membrane Potential Assay Kit Red Fluorescence, sensitive to transmembrane Potential, optimizes experimental conditions, and establishes Nav1.1 targeted toxicity determination Fluorescence detection technology of an excitation mode and an inhibition mode respectively.

The present disclosure provides a rapid screening method for marine neurotoxin fluorescence, comprising:

(1) screening for agonism: adding a compound to be detected and a negative control and a positive control into a CHO cell containing Nav1.1, and detecting by an enzyme-labeling instrument;

(2) screening for inhibition: adding a compound to be detected, a negative control and a positive control into a CHO cell containing Nav1.1, adding a given dose of agonist veratridine, and detecting by an enzyme-labeled instrument;

(3) a report is generated.

In a preferred embodiment, in step (1), the negative control is HHBS buffer and the positive control is Veratridine (Veratridine).

In a preferred embodiment, step (1) comprises:

preparing a fluorescent dye working solution;

diluting a stock solution of a compound to be detected containing the compound to be detected into a working solution to be detected;

③ culturing cells: culturing CHO cells containing Nav1.1;

removing liquid in the cell culture hole, adding a fluorescent dye working solution and an HHBS buffer solution into the cell culture hole, incubating in an incubator at 37 ℃ for 30min, and standing at room temperature for 30 min;

detecting by an enzyme labeling instrument: adding a working solution to be detected, a negative control and a positive control into the CHO cells containing Nav1.1 respectively, and detecting.

In a preferred embodiment, in step (r), the membrane potential red fluorescent dye is applied at a ratio of 1: diluting with HHBS buffer solution at a ratio of 10 to prepare fluorescent dye working solution.

The membrane potential red fluorescent dye is a slow response probe, the optical reaction amplitude of the probe is much larger than that of a fast response probe, the change of the fluorescence value is usually 1 percent per mV, and the probe is suitable for detecting the potential change caused by the ion channel activity. The principle is shown in fig. 1. After incubation with cells, the fluorescent dye is adsorbed to the outer layer of the cell membrane and binds to the quencher, and the reading of the microplate reader is the base number. Under a normal physiological state, a sodium ion channel Nav1.1 is in a closed state, when an agonist is added, the sodium ion channel is open, sodium ions flow into cells, the potential in the cells is increased, the fluorescent dye enters an inner membrane of the cells due to the electropositivity of the fluorescent dye and is far away from fluorescence quenching molecules in external liquid, the fluorescence signal is greatly improved, and the mode can be used for measuring a sodium ion channel activator; on the contrary, when the sodium ion channel inhibitor is measured, veratridine is taken as an activator to maintain the open state of the sodium channel, a strong fluorescence signal is obtained, when the inhibitor is added, the veratridine can not effectively open the ion channel, the fluorescence signal is obviously weakened, and the mode can be used for measuring the sodium ion channel inhibitor. In the excitation or inhibition mode, the rapid primary screening, the determination of the excitation/inhibition curve and the EC50/IC50 value of the toxin can be realized.

In a preferred embodiment, in the second step, the stock solution of the test compound is diluted to a suitable single-site concentration of 1 μ M to 20 μ M with HHBS buffer as the working solution to be tested.

In a preferred embodiment, in step (c), the CHO cells used are CHO cells containing Nav1.1, the gene information of Nav1.1: human nav1.1(SCN1A), NM — 001165963, CDS size 6030 bp.

In a preferred embodiment, in step (v), microplate reader parameters are set as follows: excitation light: 620 nm; light emission: 665nm, taking out the plate, adding 50 mul/hole of the working solution to be detected, detecting for 1.0min by entering the plate at an interval of 300ms, and adding the agent for detection hole by hole.

In a preferred embodiment, the stock solution of the test compound is diluted with HHBS buffer solution by 8-10 gradient concentrations step by step to serve as the test working solution for standby, and during the test, the test working solution with the gradient concentrations is sequentially tested hole by hole to draw a fluorescence-concentration excitation curve.

In a preferred embodiment, in step (2), the negative control is HHBS buffer and the positive control is tetrodotoxin.

In a preferred embodiment, step (2) comprises:

preparing a fluorescent dye working solution;

diluting veratridine stock solution with HHBS buffer solution to obtain veratridine working solution;

diluting the compound to be detected into working solution to be detected;

fourthly, cell culture: culturing CHO cells containing Nav1.1, plating for about 18h, and detecting when the cells grow to about 80%;

fifthly, removing liquid in the cell culture hole, adding a fluorescent dye working solution and an HHBS buffer solution into the cell culture hole, incubating for 30min in an incubator at 37 ℃, and then standing for 30min at room temperature;

sixth, enzyme-linked immunosorbent assay: adding a working solution to be detected, a negative control and a positive control into the CHO cell containing Nav1.1 respectively, adding a veratridine working solution, and detecting.

In the inhibition mode, veratridine open sodium ion channels need to be added in advance, so the concentration needs to be optimized.

In a preferred embodiment, in step (r), the membrane potential red fluorescent dye is applied at a ratio of 1: diluting with HHBS buffer solution at a ratio of 10 to prepare fluorescent dye working solution.

In a preferred embodiment, in step (c), the stock solution of the test compound is diluted to a suitable single-point concentration of 1 μ M to 20 μ M with HHBS buffer as the working solution to be tested.

In a preferred embodiment, in step iv, the CHO cells used are CHO cells containing nav1.1, nav1.1 gene information: human nav1.1(SCN1A), NM — 001165963, CDS size 6030 bp.

In a preferred embodiment, in step sixty, microplate reader parameters are set as follows: excitation light: 620 nm; light emission: 665nm, taking out the plate, adding working solution to be detected 20 μ l/hole, detecting for 1.0min at an interval of 300ms, taking out the plate, adding veratridine working solution 50 μ l/hole, detecting for 1.0min, adding chemicals one by one to detect

In a preferred embodiment, the stock solution of the test compound is diluted with HHBS buffer solution by 8-10 gradient concentrations step by step to serve as the test working solution for standby, and during the test, the test working solution with the gradient concentrations is sequentially tested hole by hole to draw a fluorescence-concentration inhibition curve.

The present disclosure has the following advantages:

the method is based on the CHO cell stably expressing Nav1.1 ion channel protein, adopts red fluorescent dye sensitive to transmembrane potential, optimizes experimental conditions, establishes a sodium ion channel Nav1.1 targeted toxicity determination fluorescence screening technology, can quickly perform primary screening on an agonist and an inhibitor, obtains an excitation/inhibition curve and an EC50/IC50 value, and provides a strategy for low-cost quick screening of marine neurotoxin.

Drawings

The accompanying drawings, which are included to provide a further understanding of the disclosure and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the disclosure and together with the description serve to explain the principles of the disclosure.

FIG. 1 is a schematic diagram showing the working principle of fluorescence screening of sodium ion channel targets;

FIG. 2 is a schematic diagram showing a veratridine working concentration screening real-time fluorescence plot;

figure 3 is a schematic diagram showing the activation curve of veratridine;

FIG. 4 is a schematic diagram showing the inhibition curves of tetrodotoxin;

FIG. 5 is a schematic showing the change in fluorescence of unrelated compounds in agonistic and inhibitory modes;

FIG. 6 is a primary screen schematic showing the inhibitory effect of various compounds Nav1.1;

FIG. 7 is a schematic diagram showing a preliminary screening of the agonistic effect of various compounds Nav1.1;

FIG. 8 is a graph showing the inhibition curves and IC50 values for each inhibitor;

fig. 9 is a graph showing the agonistic curves and EC50 values for each agonist.

Detailed Description

The present disclosure will be described in further detail with reference to the drawings and embodiments. It is to be understood that the specific embodiments described herein are for purposes of illustration only and are not to be construed as limitations of the present disclosure. It should be further noted that, for the convenience of description, only the portions relevant to the present disclosure are shown in the drawings.

It should be noted that the embodiments and features of the embodiments in the present disclosure may be combined with each other without conflict. The present disclosure will be described in detail below with reference to the accompanying drawings in conjunction with embodiments.

Examples

The specific experimental steps are as follows:

solution preparation:

(1)1M HEPES solution preparation: 2.38g of HEPES powder was weighed out, and ultrapure water was added thereto to adjust the volume to 10 ml.

(2) HHBS buffer preparation: 49ml of HBSS buffer (Hank's Balanced salt solution) was aspirated, and 1ml of 1M HEPES solution (final concentration 20mM) was added.

Example 1Nav1.1-CHO cell culture and preparation of experiments

The experiment used a CHO cell line that stably expressed Nav1.1 channel, Nav1.1-CHO (Nav1.1 gene information: human Nav1.1(SCN1A), NM-001165963, CDS size 6030 bp). In a cell culture dish, F-12 medium containing 10% FBS, 1% penicillin/streptomycin mixture (100X) and 200. mu.g/ml hygromycin B was placed at a constant temperature of 37 ℃ and 5% CO₂Culturing in a cell culture box. Transfer Nav1.1-CHO stably transfected cells to 96-well cell culture plates at 5X 10 per well⁴Individual cells, 96-well plates were coated overnight with PLL ahead. And (5) plating for about 18h, and detecting when the cells grow to about 80%.

Example 2 Rapid Primary Screen for Marine Neurotoxin-Primary Screen for agonist

Combining membrane potential red fluorescent dye with 1: diluting with HHBS buffer solution at a ratio of 10 to prepare a fluorescent dye working solution;

secondly, diluting each stock solution of the compound to be detected to proper single-point concentration (5 multiplied by final concentration, 500 nM-10 mu M is recommended) by using HHBS buffer solution;

thirdly, taking the 96-pore plate containing the Nav1.1-CHO cells to be detected out of the incubator, and placing the incubator at room temperature;

removing the culture medium of the original hole, adding 100 mul HHBS buffer solution into each hole, adding 100 mul fluorescent dye working solution into each hole, incubating in an incubator at 37 ℃ for 30min, and then standing at room temperature for 30 min;

detecting by an enzyme-labeling instrument, wherein the experiment parameters of the enzyme-labeling instrument are set as follows: excitation light: 620 nm/10; light emission: 665 nm/8. And (4) taking out the plate, adding 50 mu l/hole of the compound to be detected, and detecting for 1.0min at an interval of 300 ms. And (5) adding medicine one by one to detect. Negative control (HHBS buffer) and positive control (veratridine) were performed simultaneously.

Example 3 Rapid Primary Screen for Marine neurotoxins-Primary Screen for inhibitors

Combining membrane potential red fluorescent dye with 1: diluting with HHBS buffer solution at a ratio of 10 to prepare a fluorescent dye working solution;

diluting veratridine stock solution with HHBS buffer solution to obtain working solution (5 × final concentration) of 150 μ M;

thirdly, diluting each stock solution of the compound to be detected to proper single-point concentration (12.5 multiplied by final concentration, 1 mu M-20 mu M is recommended) by HHBS buffer solution;

fourthly, taking the 96 pore plate containing the Nav1.1-CHO cells to be detected out of the incubator, and placing the incubator at room temperature;

fifthly, removing the original well culture medium, adding 80 mul HHBS buffer solution into each well, adding 100 mul fluorescent dye working solution into each well, incubating for 30min in an incubator at 37 ℃, and then standing for 30min at room temperature;

sixthly, detecting by an enzyme-labeling instrument, wherein the experimental parameters of the enzyme-labeling instrument are set as follows: excitation light: 620 nm/10; light emission: 665 nm/8. Taking out the plate, adding 20 μ l/well of compound (12.5X) to be detected, detecting for 1.0min at an interval of 300ms, taking out the plate, adding 50 μ l/well of veratridine working solution, and detecting for 1.0 min. And (5) adding medicine one by one to detect. Negative controls (HHBS buffer) and positive controls (tetrodotoxin) were also performed.

Example 4 agonist Curve plotting and EC50 determination

And diluting the stock solution of the compound to be detected by HHBS buffer solution step by step for 8-10 concentrations to be used as working solution to be detected for later use. The other operations are the same as the 'agonist primary screening', and during the determination, the working solution to be determined with gradient concentration is sequentially determined hole by hole, and a fluorescence-concentration curve is drawn.

Example 5 inhibition Curve plotting and IC50 determination

And diluting the stock solution of the compound to be detected by HHBS buffer solution step by step for 8-10 concentrations to be used as working solution to be detected for later use. The other operations are the same as the 'inhibitor primary screening', and during the determination, the working solution to be determined with gradient concentration is sequentially determined hole by hole, and a fluorescence-concentration curve is drawn.

Example 6 optimization of experimental conditions:

(1) selection of fluorescent probes

The experiment selects a commercial Red fluorescent probe AAT Bioquest, Quest Membrane Potential Assay Kit Red Fluorescence and a quencher for matching use, the fluorescent dye is a slow response probe, the optical reaction amplitude of the fluorescent dye is much larger than that of a fast response probe, the change of the fluorescent value is usually 1 percent per mV, and the fluorescent dye is suitable for indicating the Potential change caused by the ion channel activity.

(2) Concentration optimization of veratridine working solution in inhibition mode

In the inhibition mode, veratridine open sodium ion channels need to be added in advance, so the concentration needs to be optimized. The real-time fluorescence changes of veratridine as an agonist at different concentrations (0. mu.M-200. mu.M) are shown in FIG. 2. The intensity and the acceleration of the fluorescence signal are gradually increased along with the increase of the veratridine concentration, and about 80% of the maximum signal position, namely 30 mu M, is finally selected as the veratridine working concentration in the experiment, so that the fluorescence signal is stronger and has sharp change, and the fluorescence signal is suitable for indicating the action effect of the inhibitor.

(3) Method verification

The agonist mode is verified by a known sodium channel agonist, veratridine.

The activation curves are shown in figure 3. The intensity of the fluorescence signal is obviously increased along with the increase of the concentration of the veratridine, and the open effect of the veratridine as an agonist on a sodium ion channel is verified, and the EC50 of the veratridine is about 24 mu M.

② inhibitor mode by known sodium ion channel inhibitor tetrodotoxin (TTX) verification.

The inhibition curves are shown in fig. 4, the opening capacity of veratridine to sodium ion channels is inhibited to different degrees in the presence of different concentrations of TTX, the higher the concentration of TTX is, the more the ion channel can not be opened, so that the fluorescence intensity is obviously reduced compared with the fluorescence intensity when veratridine is only present, and when the concentration of TTX reaches 100nM, the channel opening effect of 30 μ M veratridine is almost completely inhibited. TTX had an IC50 value of approximately 18nM, as a strong inhibitor of Nav1.1, as can be obtained from the inhibition curves.

Compound # 14 for no interaction with nav1.1 channel: scallop toxin 2(PTX 2).

The changes in fluorescence in the agonistic and inhibitory modes determined according to the method are shown in FIG. 5. In the agonistic mode, as the concentration of the compound increases, the fluorescence signal is always at the baseline level with no significant increase. In the inhibition mode, the fluorescence signal is always at the signal intensity of 30 mu M veratridine excitation with the increase of the compound concentration, and is not obviously reduced. The compound No. 14 was shown to have no interaction with the Nav1.1 channel.

Example 7 Marine neurotoxin Nav1.1 Targeted toxicity assay-Primary Screen

Using the methods established by the present disclosure, agonist and inhibitor prescreening was performed for 12 marine toxins: the 12 marine toxins are:

1 #: dcGTX2&3 (deaminoformyl gonyatoxin 2& 3);

2 #: STX (saxitoxin);

3 #: GTX1&4 (gonyautoxin 1& 4);

4 #: dcSTX (decarbamoyl saxitoxin);

5 #: GTX2&3 (gonyautoxin 2& 3);

6 #: DTX1 (finotoxin 1);

7 #: GTX6 (gonyautoxin 6);

8 #: DTX2 (finotoxin 2);

9 #: OA (okadaic acid);

10 #: gymnodinium breve toxin 1(Brevetoxin 1);

11 #: gymnodinium breve toxin 2(Brevetoxin 2);

12 #: short Euglena toxin 3(Brevetoxin 3).

First, in the inhibitor mode, using a 100nM + 30. mu.M veratridine assay system, results are shown in FIG. 6, with 1#, 2#, 3#, 4#, and 5# showing strong inhibitor effect, 7# showing weak inhibitor effect, and 6#, 8#, 9#, 10#, 11#, and 12# showing no inhibition effect.

Furthermore, the initial screening of the agonist mode was continued for 8# to 12# and the results are shown in fig. 7, with 10#, 11#, 12# showing significant agonist effect and no agonist effect for 8#, 9#, as compared to the control.

(1) Marine neurotoxin Nav1.1 Targeted toxicity assay-inhibition Curve and IC50

The results of the measurement of the inhibition curves of compounds 1#, 2#, 3#, 4#, 5#, and 7# which exhibited strong inhibitors in the preliminary screening are shown in fig. 8. Compound IC50 values showed strong and weak toxicity. Among them, GTX1&4 is the most toxic (IC50 ═ 0.439), about 40 times that of TTX; STX and dcSTX are also more toxic than TTX, and GTX2&3 is substantially equivalent to TTX.

(2) Marine neurotoxin Nav1.1 Targeted toxicity assay-activation Curve and IC50

The compounds 10#, 11#, and 12# which exhibited strong agonists in the preliminary screening were subjected to the measurement of the agonistic curve, and the results are shown in fig. 9. The EC50 value of the compound showed strong and weak toxicity. Among them, Euglenopoxin 1 showed very strong sodium channel toxicity, EC50 was 8.47 nM.

In the description herein, reference to the description of the terms "one embodiment/mode," "some embodiments/modes," "example," "specific example," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment/mode or example is included in at least one embodiment/mode or example of the application. In this specification, the schematic representations of the terms used above are not necessarily intended to be the same embodiment/mode or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments/modes or examples. Furthermore, the various embodiments/aspects or examples and features of the various embodiments/aspects or examples described in this specification can be combined and combined by one skilled in the art without conflicting therewith.

Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present application, "plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.

It will be understood by those skilled in the art that the foregoing embodiments are merely for clarity of illustration of the disclosure and are not intended to limit the scope of the disclosure. Other variations or modifications may occur to those skilled in the art, based on the foregoing disclosure, and are still within the scope of the present disclosure.