CN107163127A - Applications of the SPNP I in Nav1.4 passage tool reagents are prepared - Google Patents
Applications of the SPNP I in Nav1.4 passage tool reagents are prepared Download PDFInfo
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- CN107163127A CN107163127A CN201710572687.XA CN201710572687A CN107163127A CN 107163127 A CN107163127 A CN 107163127A CN 201710572687 A CN201710572687 A CN 201710572687A CN 107163127 A CN107163127 A CN 107163127A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
- C07K14/43518—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from spiders
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Abstract
The invention discloses a kind of application process of biologically active polypeptide in voltage-gated sodium channel tool reagent Nav1.4 types sodium channel dead-end inhibitor is prepared, described biologically active polypeptide is spider suppression sodium peptide I（SPNP‑I）, the verification experimental verification active peptides are to Nav1.4 types voltage-gated sodium channel inactivation with obvious inhibitory action.When preparing voltage-gated sodium channel hypotype tool molecule, it is 1 μm of ol/L to prepare the concentration of cell external administration.Concentration compliance is presented in suppression of the SPNP I to Nav1.4 types sodium channel, is a kind of preferable Nav1.4 types sodium channel tool reagent.
Description
Technical field
The present invention relates to a kind of purposes of active peptides in ion channel tool reagent is prepared, chemical legal system is especially used
Standby Guangxi tassel hair spider Toxic extraction thing-spider suppression sodium peptide-I（It is abbreviated as SPNP-I）Preparing the examination of voltage-gated sodium channel instrument
Purposes in the inhibitor of agent-Nav1.4 types sodium channel.
Background technology
Voltage-gated sodium channel is distributed widely in the excitabilities such as neuron, cardiac muscle, vascular smooth muscle, pancreas, skeletal muscle
A kind of important transmembrane structure albumen in tissue, its participate in regulation secretion of neurotransmitter, vessel retraction, pancreatic secretion and
One of the various physiological processes such as skeletal muscle excitability, while be also the important target of medicine effect.Sodium channel is in action electricity
Key effect in the generation and communication process of position, voltage-gated sodium channel turns into the action target of many animals and plants toxin.
The structure-function relationship research of sodium-ion channel has become international study hotspot.Voltage-gated sodium channel is likely into
For the critical treatment target spot of the diseases such as nerve and angiocarpy.The zootoxin of nature be a class have very high practical value and
The tool reagent or drug targeting material of application prospect, are not only the powerful mean that venomous animal resists natural enemy, are also to be engaged in god
Valuable materials through biology and Physiologic Studies, the exploitation of natural original new drug and protein basic research, while being also to grind
Study carefully the uniqueness " molecular probe and decipher " of ion channel.So far, from many animals（As scorpion, spider, snake and ocean are dynamic
Thing etc.）Venom in identified the active components of many batrachotoxins.They pass through the diverse location phase with sodium channel
Occur corresponding change with reference to so as to the dynamic characteristic for causing passage, such as passage de-inactivation, passage mouthful, easyization activate,
Inactivation delays.By illustrating the molecular mechanism of these specific modulator batrachotoxins, except that can go deep into theory
Speculate outside functional activity difference and the gate active mechanism between the hypotype of sodium channel, can also be that they are developed into the treatment mankind
The new drug of relevant disease provides sufficient theoretical foundation and wide application prospect.
The content of the invention
It is contemplated that preparing voltage gated sodium in providing a kind of Guangxi tassel hair spider Toxic extraction thing-spider suppression sodium peptide-I
Application in the inhibitor of passage tool reagent-Nav1.4 types sodium channel, described Guangxi tassel hair spider Toxic extraction thing-spider suppression sodium
Peptide-I（It is abbreviated as SPNP-I）, its amino acid sequence is：
NH2-Ala Cys Gly Gln Phe Trp Trp Lys Cys Gly Glu Gly Lys Pro Pro Cys Cys
Ala Asn Phe Ala Cys Lys Ile Gly Leu Tyr Leu Cys Ile Trp Ser Pro-OH,
It is characterized in that Guangxi tassel hair spider Toxic extraction thing-spider suppression sodium peptide-I is used to prepare as single effective active component
Nav1.4 types sodium channel inhibitor.
A kind of described Guangxi tassel hair spider Toxic extraction thing-spider suppression sodium peptide-I is preparing the examination of voltage-gated sodium channel instrument
Application in the inhibitor of agent-Nav1.4 types sodium channel, it is characterised in that described spider suppression sodium peptide-I the 2nd half Guang ammonia of N-terminal
Between acid and the 16th cysteine, between the 9th cysteine of N-terminal and the 21st cysteine, the 15th cysteine of N-terminal
Disulfide bond is formed respectively between the 28th cysteine.
The spider presses down sodium peptide-I and is used to prepare Nav1.4 types sodium channel inhibitor as single effective active component, with cell
The mode of external administration is prepared into Nav1.4 types sodium channel tool reagent.
Spider presses down sodium peptide-I when preparing Nav1.4 types sodium channel tool reagent or inhibitor, prepares the agent of cell external administration
Measure as 1 μm of ol/L.
Spider presses down sodium peptide-I and concentration dependent and time dependent is presented to the suppression of Nav1.4 types sodium channel, be it is a kind of compared with
Good Nav1.4 types sodium channel tool reagent.
Brief description of the drawings
Fig. 1 is influences of the SPNP-I to Nav1.4 types sodium channel.
Fig. 2 is that SPNP-I suppresses m- effect relation curve during Nav1.4 type sodium channels.
Embodiment
It is of the invention with more abundant disclosure in order to be better understood from, below will be by extracellular method of administration, using transfection
HEK293T cell models illustrate spider suppression sodium peptide-I Nav1.4 types sodium channel inhibitory action.
1st, experiment material and method
1.1 human embryonic kidney cells（HEK293T）Culture and transfection
The cell adapted sour environments of HEK293T, pH values at 6.9~7.1, can smooth adherent growth, action will when changing liquid
Gently.The general DMEM culture mediums with high sugar.
1.1.1 the Secondary Culture of cell
(1) HEK293 passages opportunity, to converge up to 80-90%, after cell is covered with 60mm culture dishes, sops up culture
Liquid.
(2) add appropriate PBS rinsings twice, sop up.
(3) add the pancreatin of 0.5mL 0.25%, shake up.Pancreatin handles 2-3min in 37 DEG C of incubators.In micro- Microscopic observation
Whether cell all takes off wall.Add appropriate 10% NBCS DMEM nutrient solutions and terminate pancreatin reaction.
(4) cell mass is gently inhaled with pipette and breaks into individual cells.Cell is averagely divided into equipped with preheating training by 1: 3
In the culture dish of nutrient solution, in 37 DEG C, 5% CO2, cultivate in 15% relative humidity incubator.
1.1.2 the liposome transfection method of cationic:
（1）In the day before transfection, for 35mm culture dishes, the nutrient solution culture of antibiotic is free of with 2mL per ware.
（2）The same day of transfection, cell density is preferably up to 80-90%, sops up old nutrient solution, and PBS is rinsed once, changes 2mL into
Serum-free opti-MEM nutrient solutions.
（3）It is 4 μ g per ware DNA consumptions, is diluted to 250 μ L respectively with serum-free serum-free opti-MEM nutrient solutions, gently
Mix, be stored at room temperature 5min.
（4）10 μ L liposomes are added in 250 μ L serum-free opti-MEM nutrient solutions simultaneously, gently mixes, is stored at room temperature
5min。
（5）The liposome of dilution is added into isometric DNA to mix, 20min is stored at room temperature.
（6）0.5mLDNA/ liposome complexes are gently mixed, are added dropwise in cell, are gently mixed, normal condition training
Support.
（7）DNA/ liposome complexes are allowed with after cells contacting 4-6h, changing the training of 10% NBCS DMEM nutrient solutions into
Support.Patch clamp experiments can be carried out in 24-72h.
1.2 patch-clamp electrophysiologic activities are tested
Patch clamp experiments are in room temperature（25±1℃）Carry out, using whole-cell patch-clamp recording technique.Select the smooth visible, born of the same parents of plasma membrane
Matter uniformly has the HEK293T cells of green fluorescence as experimental cell.Electric current record is utilized by whole-cell patch-clamp recording technique
EPC9 amplifiers (HEKA companies, German) are carried out on computers.Computer recording and analysis system use Pulse+
The softwares of Pulsefit 8.0.Vitreous electricity pole pipe is borosilicate glass capillary tube（Nanjing spring teaching experiment equipment factory）.Glass
The step of electrode two draws and formed, and eletrode tip diameter is about 3 μm after polished instrument (Narishige, Japan) polishing, charges electrode
Electrode resistance is 1-3M Ω after liquid.Patch clamp experiments will be carried out at ambient temperature, and the variation of temperature is most in whole experiment process
It is many to be no more than 2 DEG C up and down.Using the software analysis experimental results of SigmaPlot 9.0.
2nd, experimental result and analysis
Influences of 2.1 SPNP-I to Nav1.4 type voltage-gated sodium channels
With whole-cell patch-clamp recording technique, we further study sodium of the SPNP-I to transient expression in HEK293T cells and lead to
Road hypotype Nav1.4 inhibitory action.Sodium channel hypotype electric current is induced in same depolarising mode：Cell membrane potential is clamped down on
In -100 mV, a test voltage -10mV is given with 20 ms time-histories, is repeated once every 5 s.
As shown in figure 1,1 μM of SPNP-I has certain inhibitory action to the inactivation of Nav1.4 passages, can substantially it delay
The inactivation of Nav1.4 sodium channels, its inhibition level is 72%.SPNP-I is to the suppression of Nav1.4 passages or delays deactivation to present
Obvious concentration compliance, SPNP-I half useful effect dosage IC50For 340 nmol/L.SPNP-I is to Nav1.4 sodium channels
Inactivation suppress have time dependent, when concentration increase be 1 μM when, maximum suppression journey can be reached within about 2 min time
Degree（See Fig. 2）.
SEQUENCE LISTING
<110>Changsha Qin Cai bio tech ltd
<120>Spider suppression sodium peptide-I
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 33
<212> PRT
<213>Guangxi tassel hair spider
<400> 1
Asp Cys Leu Gly Leu Phe Trp Ile Cys Gln Tyr Met Asp Asp Lys
1 5 10 15
Cys Cys Ala Asn Phe Ala Cys Lys Ile Gly Leu Tyr Leu Cys Ile
16 20 25 30
Trp Ser Pro
31
Claims (5)
1. a kind of Guangxi tassel hair spider Toxic extraction thing-spider suppression sodium peptide-I is preparing sodium channel tool reagent-Nav1.4 sodium channels
Application in inhibitor, described Guangxi tassel hair spider Toxic extraction thing-spider suppression sodium peptide-I（It is abbreviated as SPNP-I）, its amino acid
Sequence is：
NH2-Ala Cys Gly Gln Phe Trp Trp Lys Cys Gly Glu Gly Lys Pro Pro Cys Cys
Ala Asn Phe Ala Cys Lys Ile Gly Leu Tyr Leu Cys Ile Trp Ser Pro-OH,
It is characterized in that Guangxi tassel hair spider Toxic extraction thing-spider suppression sodium peptide-I is used to prepare as single effective active component
Nav1.4 types sodium channel inhibitor.
2. application process according to claim 1, it is characterised in that spider suppression sodium peptide-I is used as single effective active component
For preparing Nav1.4 types sodium channel dead-end inhibitor.
3. application process according to claim 1, it is characterised in that spider suppression sodium peptide-I is prepared into cell external administration
Nav1.4 types sodium channel tool reagent.
4. application process according to claim 1, it is characterised in that spider presses down sodium peptide-I and preparing Nav1.4 types sodium channel
When tool reagent or dead-end inhibitor, it is 1 μm of ol/L to prepare the dosage of cell external administration.
5. application process according to claim 1, it is characterised in that spider suppression sodium peptide-I to Nav1.4 types sodium channel
Suppress deactivation and concentration dependent and time dependent is presented.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710572687.XA CN107163127A (en) | 2017-07-14 | 2017-07-14 | Applications of the SPNP I in Nav1.4 passage tool reagents are prepared |
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| CN201710572687.XA CN107163127A (en) | 2017-07-14 | 2017-07-14 | Applications of the SPNP I in Nav1.4 passage tool reagents are prepared |
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| CN107163127A true CN107163127A (en) | 2017-09-15 |
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| CN201710572687.XA Pending CN107163127A (en) | 2017-07-14 | 2017-07-14 | Applications of the SPNP I in Nav1.4 passage tool reagents are prepared |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1473851A (en) * | 2003-08-06 | 2004-02-11 | 湖南师范大学 | Jingzhao chilobrachys spider toxin |
-
2017
- 2017-07-14 CN CN201710572687.XA patent/CN107163127A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1473851A (en) * | 2003-08-06 | 2004-02-11 | 湖南师范大学 | Jingzhao chilobrachys spider toxin |
Non-Patent Citations (2)
| Title |
|---|
| XIAO,Y.: "Characterization of the excitatory mechanism", 《TOXICON》 * |
| YUCHENG XIAO: "Jingzhaotoxin-I, a Novel Spider Neurotoxin Preferentially", 《J.BIOL.CHEM.》 * |
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