CN105879011A - Application of polypeptide to preparation of Nav1.7 sodium channel modulation preparation - Google Patents
Application of polypeptide to preparation of Nav1.7 sodium channel modulation preparation Download PDFInfo
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- CN105879011A CN105879011A CN201610249727.2A CN201610249727A CN105879011A CN 105879011 A CN105879011 A CN 105879011A CN 201610249727 A CN201610249727 A CN 201610249727A CN 105879011 A CN105879011 A CN 105879011A
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Abstract
The invention discloses a method for applying a biological activity polypeptide to preparation of a voltage-gated channel tool reagent which is a Nav1.7 sodium channel inhibitor. The bioactive polypeptide is spider inhibiting natriuretic peptide-IX (SPNP-IX), and it is verified through tests that the bioactive polypeptide has an obvious inhibiting effect on a Nav1.7 voltage-gated sodium channel. When the voltage-gated sodium channel subtype tool reagent is prepared, the prepared extracellular drug administration concentration is 1 [mu] mol/L, and the median effective dose IC50 of the SPNP-IX is 450 nmol/L. The inhibition to the Nav1.7 sodium channel by the SPNP-IX has time dependence, and the SPNP-IX is a good Nav1.7 sodium channel tool reagent.
Description
Technical field
The present invention relates to a kind of active polypeptide purposes in ion channel tool reagent, especially press down sodium peptide-IX(be abbreviated as SPNP-IX with Guangxi tassel hair spider Toxic extraction thing-Aranea prepared by chemical method) purposes in preparing voltage-gated sodium channel tool reagent-Nav1.7 type sodium channel inhibitor.
Background technology
Voltage-gated sodium channel is distributed widely in the excitability tissues such as neuron, cardiac muscle, vascular smooth muscle, pancreas, skeletal muscle, it is a kind of important transmembrane structure albumen, it participates in the various physiological processes such as regulation secretion of neurotransmitter, vasoconstriction, pancreatic secretion and skeletal muscle irritability, is also one of the important target of medicine effect simultaneously.The sodium channel key effect in the generation and communication process of action potential, voltage-gated sodium channel becomes the action target of a lot of animals and plants toxin.The structure-function relationship research of sodium-ion channel has become as international study hotspot.Voltage-gated sodium channel is likely to become the critical treatment target spot of the disease such as nerve and cardiovascular.The zootoxin of nature is that a class has the highest practical value and the tool reagent of application prospect or drug targeting material, it is not only venomous animal and resists the powerful mean of natural enemy, also it is to be engaged in neurobiology and Physiologic Studies, the exploitation of natural original new drug and the valuable materials of protein basic research, is the most also the uniqueness " molecular probe and decipher " of research ion channel.Up to now, from the venom of many animals (such as scorpion, Aranea, Serpentis and marine animal etc.), identified the active component of a lot of batrachotoxin.They by combine with the diverse location of sodium channel thus cause passage dynamic characteristic occur corresponding change, as passage de-inactivation, passage mouth, easily change activation, inactivate delay.By illustrating the molecular mechanism of these specificity modulator batrachotoxins, in addition to can deeply speculating the difference of the functional activity between the hypotype of sodium channel and gate active mechanism in theory, it is also possible to provide sufficient theoretical basis and wide application prospect for they being developed into the new drug for the treatment of mankind's relevant disease.
Summary of the invention
It is contemplated that in providing a kind of Guangxi tassel hair spider Toxic extraction thing-Aranea to press down sodium peptide-IX in the application preparing voltage-gated sodium channel tool reagent-Nav1.7 type sodium channel inhibitor, described Guangxi tassel hair spider Toxic extraction thing-Aranea presses down sodium peptide-IX(and is abbreviated as SPNP-IX), its aminoacid sequence is:
NH2-Glu Cys Thr Lys Leu Leu Gly Gly Cys Thr Lys Asp Ser Glu Cys
Cys Pro
His Leu Gly Cys Arg Lys Lys Trp Pro Tyr His Cys Gly Trp Asp Gly Thr Phe-NH2 ,
It is characterized in that Guangxi tassel hair spider Toxic extraction thing-Aranea presses down sodium peptide-IX as single effective active component for preparing Nav1.7 type sodium channel inhibitor.
Described Aranea presses down between the 2nd cysteine of N end and the 16th cysteine of sodium peptide-IX, between the 9th cysteine of N end and the 21st cysteine, forms disulfide bond respectively between the 15th cysteine of N end and the 29th cysteine.
This Aranea presses down sodium peptide-IX as single effective active component for preparing Nav1.7 type sodium channel inhibitor, is prepared as Nav1.7 type sodium channel tool reagent in the way of the administration of extracellular.
Aranea presses down sodium peptide-IX preparing Nav1.7 type sodium channel tool reagent or during inhibitor, and the dosage that preparation extracellular is administered is 1 μm ol/L.
Aranea presses down the half useful effect dosage IC of sodium peptide-IX suppression Nav1.7 type sodium channel50It is 110
nmol/L。
Aranea presses down the sodium peptide-IX suppression presentative time compliance to Nav1.7 type sodium channel, is a kind of preferably Nav1.7 type sodium channel tool reagent.
Accompanying drawing explanation
Fig. 1 is that 1 μm ol/L SPNP-IX is on the impact of Nav1.7 type sodium channel on rat drg neuron.
Fig. 2 is SPNP-IX m-effect relation curve when acting on rat DRG cell Nav1.7 sodium channel.
Fig. 3 is concentration-effect relation curve that SPNP-IX acts on rat DRG cell Nav1.7 sodium channel.
Fig. 4 is the SPNP-IX impact on rat DRG cell Nav1.7 sodium channel current-voltage relationship.
Fig. 5 is the SPNP-IX dynamic (dynamical) impact of Steady-state inactivation on rat DRG cell Nav1.7 sodium channel.
Detailed description of the invention
In order to be better understood from and more fully disclose the present invention, transfection HEK293T cell model will be used to illustrate that Aranea presses down the Nav1.7 type sodium channel inhibitory action of sodium peptide-IX by extracellular route of administration below.
1
, experiment material and method
The cultivation of 1.1 HEKC (HEK293T) and transfection
The cell adapted sour environment of HEK293T, pH value when 6.9～7.1, can adherent growth smoothly, when changing liquid, action wants light.The general DMEM culture medium with high sugar.
1.1.1 the Secondary Culture of cell
(1) HEK293 passage converges for reaching 80-90% opportunity, after cell covers with in 60mm culture dish, sops up culture fluid.
(2) add appropriate PBS to rinse twice, sop up.
(3) add 0.5mL 0.25% pancreatin, shake up.In 37 DEG C of incubators, pancreatin processes 2-3min.Wall is the most all taken off in basis of microscopic observation cell.Add appropriate 10% new-born calf serum DMEM culture fluid and terminate pancreatin reaction.
(4) with pipet cell mass inhaled gently and break into individual cells.Cell is averagely divided in the culture dish equipped with preheating culture fluid by 1: 3, in 37 DEG C, 5% CO2, 15% relative humidity incubator is cultivated.
1.1.2 the liposome transfection method of cationic:
(1) at day before transfection, for 35mm culture dish, every ware 2mL culture fluid without antibiotic is cultivated.
(2) on the same day transfected, cell density is preferably up to 80-90%, sops up old culture fluid, and PBS rinses once, changes 2mL serum-free opti-MEM culture fluid into.
(3) every ware DNA consumption is 4 g, is diluted to 250 L respectively with serum-free serum-free opti-MEM culture fluid, mixes gently, and room temperature stands 5min.
(4) being added by 10 L liposomees in 250 L serum-free opti-MEM culture fluid simultaneously, mix gently, room temperature stands 5min.
(5) liposome of dilution being added equal-volume DNA mixing, room temperature stands 20min.
(6) being mixed gently by 0.5mLDNA/ liposome complex, be added dropwise in cell, mix gently, normal condition is cultivated.
(7) allow after DNA/ liposome complex and cells contacting 4-6h, change 10% new-born calf serum DMEM culture fluid into and cultivate.Patch clamp experiments can be carried out in 24-72h.
1.2 patch-clamp electrophysiologic activity experiments
Patch clamp experiments is all carried out in room temperature (25 ± 1 DEG C), uses whole-cell patch-clamp recording technique.Select smooth visible, the kytoplasm of plasma membrane and have the HEK293T cell of green fluorescence uniformly as experimental cell.Electric current record utilizes EPC9 amplifier by whole-cell patch-clamp recording technique
(HEKA company, German) is carried out on computers.Computer recording and the system of analysis use Pulse+Pulsefit
8.0 software.Glass electrode pipe is borosilicate glass capillary tube (Nanjing spring water education experiment equipment factory).Glass electrode two step draws and forms, and polished instrument (Narishige, Japan) polishing rear electrode tip diameter is about 3 m, and charging electrode solution rear electrode resistance is 1-3M Ω.Patch clamp experiments to be carried out at ambient temperature, and in whole experimentation, the variation of temperature is the most up and down less than 2 DEG C.Use SigmaPlot 9.0 software analysis experimental result.
2, experimental result and analysis
2.1 SPNP-IX impact on Nav1.7 type voltage-gated sodium channel
Use whole-cell patch-clamp recording technique, we have studied SPNP-IX to transient expression in the inhibitory action of sodium channel hypotype Nav1.7 of HEK293T cell.Sodium channel hypotype electric current is induced in same depolarization mode: is clamped down on by cell membrane potential at-80 mV, gives a test voltage-10mV with the time-histories of 50 ms, be repeated once every 5 s.
As it is shown in figure 1,1 μM of SPNP-IX has certain inhibitory action to Nav1.7 passage, its suppression degree is respectively 83.2 ± 4.3%(n=6).SPNP-IX presents obvious time dependent to the inhibitory action of Nav1.7 passage, and when toxin concentration is 1 μM, the about time at about 1 min reaches maximum suppression.The time constant of 1 μM of SPNP-IX suppression Nav1.7 passage is 15.8 s(Fig. 2).As it is shown on figure 3, SPNP-IX presents obvious concentration compliance, half effective inhibition concentration (IC to the inhibitory action of Nav1.7 passage50) it is 110 nM.When dose curve is with Hill formula fitting, can draw the Hill constant of the concentration-response curve of SPNP-IX suppression Nav1.7 passage, its size is 0.85.
2.2
The SPNP-IX impact on Nav1.7 type voltage-gated sodium channel state of activation
Under whole-cell voltage-clamp pattern, being clamped down on by cell when-100 mV, give a series of test voltage respectively, its excursion is-80～+60
MV, the persistent period is 50 ms, and stride is+10
MV, we have detected the SPNP-IX impact on Nav1.7 passage I-V curve further, probe into its impact on sodium channel hypotype activation process, and result is as shown in Figure 4.Under the conditions of blank, when cell membrane potential is clamped down at-100 mV,
The initial activation voltage of Nav1.7 passage is-40 mV, and it is-20 that maximum current peak activates voltage
mV.SPNP-IX is added at cell peripheral, after toxin and cytosis 3 min, again with identical depolarization pulse induction I-V curve, found that SPNP-IX can make the initial activation potential of Nav1.7 sodium channel and peak point current activate voltage to direction of depolarization drift about+10
MV, but reversal potential change is inconspicuous, illustrates that toxin does not change, with the interaction of passage, the selectivity that passage is penetrating to ion.
2.3
The SPNP-IX impact on Nav1.7 type voltage-gated sodium channel Steady-state inactivation
We use a standard dipulse stimulation mode, have further looked at the SPNP-IX impact on Nav1.7 passage Steady-state inactivation feature.Result such as Fig. 5, for Nav1.7 passage, under collating condition, V1/2For-77.3 ± 1.4 mV(n=8), after adding SPNP-IX, V1/2Become-79.7 ± 1.2 mV(n=7).Result shows SPNP-IX Steady-state inactivation feature almost no impact on hNav1.7, and the most substantially changes the slope of Nav1.7 passage control curve.
SEQUENCE
LISTING
<110>
Qin Cai bio tech ltd, Changsha
<120>
Aranea presses down sodium peptide-IX
<160>
1
<170>
PatentIn version 3.3
<210>
1
<211>
35
<212>
PRT
<213>
SPNP-IX
<400>
1
Glu Cys Thr
Lys Leu Leu Gly Gly Cys Thr Lys Asp Ser Glu Cys
1
5 10
15
Cys Pro His
Leu Gly Cys Arg Lys Lys Trp Pro Tyr His Cys Gly
16
20
25
30
Trp Asp Gly
Thr Phe
31
35
Claims (7)
1. Guangxi tassel hair spider Toxic extraction thing-Aranea presses down sodium peptide-IX application in preparing voltage-gated sodium channel tool reagent-Nav1.7 type sodium channel inhibitor, described Guangxi tassel hair spider Toxic extraction thing-Aranea presses down sodium peptide-IX(and is abbreviated as SPNP-IX), its aminoacid sequence is:
NH2-Glu Cys Thr Lys Leu
Leu Gly Gly
Cys Thr Lys
Asp Ser Glu Cys Cys Pro
His Leu Gly Cys Arg
Lys Lys Trp
Pro Tyr His Cys Gly Trp Asp Gly
Thr Phe-NH2 ,
It is characterized in that Guangxi tassel hair spider Toxic extraction thing-Aranea presses down sodium peptide-IX as single effective active component for preparing Nav1.7 type sodium channel inhibitor.
A kind of Guangxi the most according to claim 1 tassel hair spider Toxic extraction thing-Aranea presses down sodium peptide-IX application in preparing voltage-gated sodium channel tool reagent-Nav1.7 type sodium channel inhibitor, it is characterized in that, described Aranea presses down between the 2nd cysteine of N end and the 16th cysteine of sodium peptide-IX, between the 9th cysteine of N end and the 21st cysteine, between the 15th cysteine of N end and the 29th cysteine, form disulfide bond respectively.
Application process the most according to claim 1 and 2, it is characterised in that Aranea presses down sodium peptide-IX as single effective active component for preparing Nav1.7 type sodium channel inhibitor.
Application process the most according to claim 1 and 2, it is characterised in that Aranea presses down sodium peptide-IX and is prepared as the Nav1.7 type sodium channel tool reagent that extracellular is administered.
Application process the most according to claim 1 and 2, it is characterised in that Aranea presses down sodium peptide-IX preparing Nav1.7 type sodium channel tool reagent or during inhibitor, and the dosage that preparation extracellular is administered is 1 μm ol/L.
Application process the most according to claim 1 and 2, it is characterised in that Aranea presses down the half useful effect dosage IC of sodium peptide-IX50It is 110 nmol/L.
Application process the most according to claim 1 and 2, it is characterised in that it is temporal dependence that Aranea presses down the effect of sodium peptide-IX.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610249727.2A CN105879011A (en) | 2016-04-20 | 2016-04-20 | Application of polypeptide to preparation of Nav1.7 sodium channel modulation preparation |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610249727.2A CN105879011A (en) | 2016-04-20 | 2016-04-20 | Application of polypeptide to preparation of Nav1.7 sodium channel modulation preparation |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1872878A (en) * | 2006-06-22 | 2006-12-06 | 湖南师范大学 | Jingzhao toxin V |
| CN104768568A (en) * | 2012-05-18 | 2015-07-08 | 詹森生物科技公司 | Huwentoxin-iv variants and methods of use |
| CN104892745A (en) * | 2015-06-16 | 2015-09-09 | 湖南理工学院 | Preparation method of Hainantoxin-III |
-
2016
- 2016-04-20 CN CN201610249727.2A patent/CN105879011A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1872878A (en) * | 2006-06-22 | 2006-12-06 | 湖南师范大学 | Jingzhao toxin V |
| CN104768568A (en) * | 2012-05-18 | 2015-07-08 | 詹森生物科技公司 | Huwentoxin-iv variants and methods of use |
| CN104892745A (en) * | 2015-06-16 | 2015-09-09 | 湖南理工学院 | Preparation method of Hainantoxin-III |
Non-Patent Citations (4)
| Title |
|---|
| MEICHUN DENG等: "Jingzhaotoxin-IX, a novel gating modifier of both sodium and potassium channels from Chinese tarantula Chilobrachys jingzhao", 《NEUROPHARMACOLOGY》 * |
| ZHI LIAO等: "Proteomic and peptidomic analysis of the venom from Chinese tarantula Chilobrachys jingzhao", 《PROTEOMICS》 * |
| 季鑫: "Nav1.7在宫颈癌中的表达及临床意义", 《万方 中国学位论文全文数据库》 * |
| 陈再冉: "虎纹捕鸟蛛毒素-Ⅳ突变体K27A的化学合成与性质以及两种海南敬钊缨毛蛛毒素的结构与功能研究", 《中国优秀博硕士学位论文全文数据库 (硕士) 基础科学辑》 * |
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Application publication date: 20160824 |