CN1186354C - Jingzhao chilobrachys spider toxin - Google Patents
Jingzhao chilobrachys spider toxin Download PDFInfo
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- CN1186354C CN1186354C CNB031247180A CN03124718A CN1186354C CN 1186354 C CN1186354 C CN 1186354C CN B031247180 A CNB031247180 A CN B031247180A CN 03124718 A CN03124718 A CN 03124718A CN 1186354 C CN1186354 C CN 1186354C
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Abstract
The present invention a Jingzhao chilobrachys spider toxin. The Jingzhao chilobrachys spider toxin-I (JZTX-I) which is obtained by the separation of Jingzhao chilobrachys spider toxin is a spider peptide molecule which comprises 33 amino acid residues, 6 cysteines exist as 3 pairs of disulfide bonds and with molecular weight of 3675.64. The extraction method of the JZTX-I toxin comprises the steps: toxin is collected from living bodies of trapped spiders by hose bundles firstly; double separation of coarse toxin is carried out by cationic exchange and inverse HPLC chromatography. An organ level test and a molecular level test on JZTX-I toxin show that the toxin is a kind of exciting spider toxin with sensitivity to nerves, muscles and hearts. The toxin does not suppress the magnitude of sensitive sodium current in sodium ion channel, but can delay the deactivating time of sodium current. The toxin is a special toxin for delaying the deactivating time of sodium current.
Description
Technical field:
The present invention relates to a kind of spider venom, be specifically related to a kind of Jingzhao chilobrachys spider toxin.
Background technology:
Often contain abundant bioactive molecule in the crude venom of the spider, but they can optionally act on the ionic channel on the excitability cytolemma, as sodium channel, calcium channel and potassium channel, thus the normal conduction of change organ level impulse of excitation.Have and to be developed to the new drug application prospect that helps human health.As, brave line analgesia peptide-I can block the N-type calcium channel of presynaptic membrane, has very significantly analgesic effect.Sodium-ion channel is the key foundation that produces action potential, is controlling the formation and the conduction of action potential on nerve trunk and muscle of depolarize phase.Have been found that the spider venom of multiple energy batrachotoxin at present, and be divided into two major types: inhibition type and excitatory.Inhibition type toxin can be blocked the opening of sodium channel, suppresses the sodium channel outward and closes inactivation and the excitatory toxin can partly suppress the sodium current size.But do not report also that so far separation and purification is a kind of to the influential spider venom of heart.
Summary of the invention:
The present invention separates acquisition one novel spider peptide quasi-molecule and other known peptide from the thick poison of China's new species of spider Jingzhao chilobrachys spider (Chilobrachys jingzhao) do not have homology, and phrenic nerve diaphragm and isolated frog heart are had tangible hormesis.
The foregoing invention purpose realizes by following scheme:
(English name jinzhaotoxin-I, aminoacid sequence JZTX-I) is the Jingzhao chilobrachys spider toxin I: Ala Cys Gly Gln Phe Trp Trp Lys Cys Gly Gln Gly Lys
5 10Pro?Pro?Cys?Cys?Ala?Asn?Phe?Ala?Cys?Lys?Ile?Gly?Leu?Tyr?Leu?Cys
15 20 25Ile?Trp?Ser?Pro。30 33
Below in conjunction with accompanying drawing this research is described in further detail.
Description of drawings:
Fig. 1 is the separation and purification collection of illustrative plates of Jingzhao chilobrachys spider toxin I (JZTX-I)
Wherein 1-A is the ion-exchange collection of illustrative plates, and ※ represents that purpose peak, 1-B are the reversed-phase HPLC collection of illustrative plates first time, and arrow represents that peak, purpose toxin place, 1-C are the reversed-phase HPLC collection of illustrative plates second time of purpose toxin
Fig. 2 is that the mass spectrum of Jingzhao chilobrachys spider toxin I (JZTX-I) is identified
Fig. 3 is the one-level chemical structure of Jingzhao chilobrachys spider toxin I (JZTX-I)
Fig. 4 is that Jingzhao chilobrachys spider toxin I (JZTX-I) is to the mouse phrenic nerve diaphragm
Wherein 4-A is that electrical stimulation adds 10
-5G/ml Jingzhao chilobrachys spider toxin I (JZTX-I), add 15 μ M tubocurarines (dTc) when 4-B is electrical stimulation, stimulus of direct current muscle adds 10 again
-5G/ml Jingzhao chilobrachys spider toxin I (JZTX-I)
Fig. 5 is the influence of Jingzhao chilobrachys spider toxin I (JZTX-I) toad cardiomotility
Fig. 6 is the influence of Jingzhao chilobrachys spider toxin I (JZTX-I) to rat dorsal root ganglion cellular sodium passage
Wherein 6-A be toxin to influence, the 6-B of two kinds of voltage-gated sodium channels of DRG cell is concentration-response curves (DRG is a rat Dorsal root nerve) that toxin influences DRG cell TTX responsive type sodium channel
Fig. 7 is that Jingzhao chilobrachys spider toxin I (JZTX-I) is to Na
v1.5 the influence of passage
Wherein 7-A is that toxin delays Nav1.5 sodium channel inactivation phase, 7-B is the concentration compliance influence of toxin to the Nav1.5 sodium channel
The Jingzhao chilobrachys spider toxin I can extract in the toxin by Hainan Jingzhao chilobrachy spider, and concrete extracting method is as follows:
1, the acquisition method of venom
With 3-4 root length is that 4 centimetres, internal diameter are that 1.5 millimeters transparent plastic hose is tied into a branch of as luring thing, clamp the chest of Hainan Jingzhao chilobrachys spider from the side with big tweezers, spider is just opened the chela pawl, at this moment luring thing to send into hose bundle stings under the pawl, it promptly embraces hose bundle with pedipalps, effectively thrusts the chela pawl in the flexible pipe and penetrates venom.Owing to sting pawl and do not penetrate poison usually simultaneously for two of every spider, and each chela pawl not necessarily penetrates whole venom, thereby said process need carry out repeatedly at every turn.Penetrate the poison back and slowly extract hose bundle under the pawl out, venom is wherein extracted out, behind frost drying, can obtain the slightly dry powder of light golden rod yellow, be thick poison with microsyringe from stinging.
The meaning of spider venom acquisition method:
Spider is because individual little toxic amount is few, and its venom collection is very difficult.The tradition method of gathering venom directly exsomatizes malicious capsule often from the live body spider, prick the Kong Houyong water dissolution with its homogenate or on malicious capsule and collect contained venom.This method exists significantly not enough, whole process is not only very loaded down with trivial details, and to obtain the productive rate of toxin not high, and the product that obtains so not only is spider excretory venom under natural situation, also contain a large amount of ripe toxin precursor protein and non-toxin composition, bring inconvenience can for the wherein separation and purification of single toxin component, main is that the live body spider also can die after having collected toxin, causes a large amount of wastes of resource.This collection spider venom method has not only solved above-mentioned difficulties well, and the live body spider can not cause death in the process of gathering venom, and can proceed artificial breeding, the utilization of repeatedly gathering venom.
2, the separation purification method of Jingzhao chilobrachys spider toxin I
Carry out secondary separation according to charged character of protein and proteinic hydrophobicity.In conjunction with cationic exchange and reversed-phase HPLC chromatography.5 milligrams of above-mentioned thick poison are dissolved in 6 milliliters of distilled waters, last sample is in advance with in the good Waters Protein-Pak CM 8HR cationic exchange coloum (5 * 50 millimeters) of 0.2 mol disodium-hydrogen and SODIUM PHOSPHATE, MONOBASIC (pH6.25) balance, with 0%-90% sodium-chlor gradient elution, time is 80 minutes, flow velocity 3 ml/min.Under detecting, the 280nm ultraviolet wavelength obtains 9 absorption peaks altogether.Collect after the 6th peak and the desalting treatment further reversed-phase HPLC purifying.The anti-phase analytical column of C18 is through 0%-50% acetonitrile (containing 0.1% trifluoroacetic acid) solution gradient wash-out, and the time is 40 minutes, and flow velocity is 0.7 ml/min.Under detecting, the 280nm ultraviolet wavelength obtains 6 absorption peaks altogether.Carry out the reversed-phase HPLC second time under the same conditions after collecting the 5th peak, obtain single purpose peak (Fig. 1), mass spectrograph detects to one-component and to record its accurate molecular weight be 3675.64 (Fig. 2).
The Jingzhao chilobrachys spider toxin I obtains its aminoacid sequence through the 491-A sequenator, is made up of 33 amino-acid residues, contains 6 halfcystines.Its in order the theoretical molecular that calculates of analytical results surveyed molecular weight by 3675 with mass spectrograph and fitted like a glove, prove the exactness of sequence, and to prove conclusively 6 halfcystines through partial reduction modification desmoenzyme solution be that form with 3 pairs of disulfide linkage exists (Fig. 3).The physicochemical property of this toxin are: purified lyophiled powder is the loose body of white or off-white color, and odorlessness very easily is dissolved in water, and the aqueous solution is bordering on achromatism and clarity.This proteinic uv-absorption maximum wavelength is 280nm, and iso-electric point (pI) is 8.34, slightly meta-alkalescence.
In order to illustrate and disclose the effect of this lps molecule better, we set forth the toxicity mechanism of Jingzhao chilobrachys spider toxin I at organ level and molecular level.
The Jingzhao chilobrachys spider toxin I is tested the influence of animal isolated organ:
1, main raw and instrument
The Jingzhao chilobrachys spider toxin I separates from the thick poison of Hainan Jingzhao chilobrachy spider as stated above and obtains, and has identified that by HPLC and MALDI TOF mass spectrograph its purity is more than 99% simultaneously; Test mice (Kunming small white mouse): the 15-20 grammes per square metre is provided by this laboratory rearing; Toad is buied on the local market, the 50-60 grammes per square metre;
Electrical stimulator: adopt home made article (teaching multipurpose electronic stimulator, model is DOC-III) RM6240B bio signal register system: adopt home made article.
2, test-results
2.1, to the influence of mouse phrenic nerve diaphragm
The mouse phrenic nerve diaphragm separates decorticating according to a conventional method.Sample after the separation gives the contraction that diaphram is induced in electricity irritation after the Tai Shi body lotion that continues to fill 95% oxygen leaves standstill 10 minutes, blank is not have obviously decay in one hour.Excite nerve and add 10
-5Behind the grams per liter spider poison, the contraction intensity of diaphram has increased by 200% behind the 5min.Another organizes sample, excites nerve to cause after the muscle excitement and shrink with 15 micromoles per liter D-tubocurarines blocking-up diaphram earlier, directly causes contraction with the electricity irritation diaphram again, add the spider poison of same concentrations after, the interior muscle excitement of 10min intensity has increased by 150% (Fig. 4).Intensity of electric stimulus 4.5V, the wide 0.2ms of ripple, frequency 15 times/minute.
2, to the influence of toad heart
The toad isolated frog heart all separates decorticating according to a conventional method.The oneself that can produce rhythmicity with the neural frog heart under the situation of blank that exsomatizes is not beaten.Add 10
-5Promptly can be observed the intensity enhancing that the frog heart shrinks behind the grams per liter spider poison in the 1min, and contraction frequency increases (Fig. 5).
Test-results shows that the Jingzhao chilobrachys spider toxin I is a kind of novel excitability spider venom, all has susceptibility to nerve, muscle, heart in sum.
The Jingzhao chilobrachys spider toxin I is to the analytical test of sodium-ion channel effect:
Main raw and instrument
The Jingzhao chilobrachys spider toxin I separates from the thick poison of Hainan Jingzhao chilobrachy spider as stated above and obtains, and has identified that by HPLC and MALDITOF mass spectrograph its purity is more than 99% simultaneously;
The test rat: this chamber is raised and is provided
Xenopus laevis: this chamber is raised and is provided
All reagent are all available from Sigma company;
Patch clamp amplifier: the German HEKA EPC9 of company;
Voltage clamp amplifier: the GeneClamp500B of U.S. AXON company.
2, test method and result
Can sodium-ion channel has 9 different hypotypes on different histoorgans, according to being divided into two big classes by tetraodotoxin (TTX) blocking-up: TTX responsive type sodium channel and TTX non-sensitive type sodium channel.Both activate with current characteristic at voltage dependent and have notable difference: in the resting potential level, the activation threshold of TTX responsive type sodium channel will be lower than TTX non-sensitive type sodium channel; Electric current complete deactivation in 2ms that the back produces is activated in TTX responsive type sodium channel, and TTX non-sensitive type sodium channel current can complete deactivation in 10ms yet.
Three kinds of voltage-sensitive type sodium channels (Nav1.7, Nav1.8 and Nav1.9) are arranged on the rat dorsal root ganglion cell, and first kind is TTX responsive type sodium channel, and second and the third be TTX non-sensitive type sodium channel.Experiment shows, under the concentration of 1 μ M, the Jingzhao chilobrachys spider toxin I does not have obvious influence to TTX non-sensitive type sodium channel, do not suppress TTX responsive type sodium current size, but can delay its inactivation time, the amplitude that delays electric current accounts for 34 ± 4% of total current size, and it is 0.995 ± 0.020 μ M (Fig. 6) that effective half influences concentration.
Nav1.5 is a TTX non-sensitive type sodium channel hypotype that is distributed on heart, the skeletal muscle cytolemma, can not be blocked by the TTX of lower concentration.The Jingzhao chilobrachys spider toxin I is similar to influence to the neuron membrane sodium channel to the influence mode of this sodium channel, but sensitivity wants high.Under the concentration of 0.167 μ M, the Jingzhao chilobrachys spider toxin I can delay sodium current 25 ± 3% inactivations, does not suppress the size of sodium current, and it is 0.300 ± 0.002 μ M (Fig. 7) that its effective half influences concentration.
Above-mentioned test-results shows that the Jingzhao chilobrachys spider toxin I does not suppress responsive type sodium current size, but can delay the sodium current inactivation time, is the toxin that a species specificity delays the sodium current inactivation time.
The aminoacid sequence of Jingzhao chilobrachys spider toxin I is as follows: Ala Cys Gly Gln Phe
5Trp?Trp?Lys?Cys?Gly?Gln?Gly?Lys?Pro?Pro?Cys?Cys?Ala?Asn?Phe
10 15 20Ala?Cys?Lys?Ile?Gly?Leu?Tyr?Leu?Cys?Ile?Trp?Ser?Pro。
25 30 33
Claims (1)
1, a kind of Jingzhao chilobrachys spider toxin, it is characterized in that the δ-Jingzhao chilobrachys spider toxin I of separation and purification from the thick poison of Jingzhao chilobrachys spider, the aminoacid sequence of this toxin is as follows: Ala Cys Gly Gln Phe Trp Trp Lys Cys Gly Gln Gly Lys Pro ProCys Cys Ala Asn Phe Ala Cys Lys Ile Gly Leu Tyr Leu Cys IleTrp Ser Pro.
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| CNB031247180A CN1186354C (en) | 2003-08-06 | 2003-08-06 | Jingzhao chilobrachys spider toxin |
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| CNB031247180A CN1186354C (en) | 2003-08-06 | 2003-08-06 | Jingzhao chilobrachys spider toxin |
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| CN1186354C true CN1186354C (en) | 2005-01-26 |
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100429229C (en) * | 2006-06-22 | 2008-10-29 | 湖南师范大学 | Jingzhao toxin V |
| CN107163125A (en) * | 2017-07-14 | 2017-09-15 | 长沙沁才生物科技有限公司 | Applications of the SPNP I in Nav1.2 passage tool reagents are prepared |
| CN107226851A (en) * | 2017-07-14 | 2017-10-03 | 长沙沁才生物科技有限公司 | Applications of the SPNP I in Nav1.5 passage tool reagents are prepared |
| CN107176986A (en) * | 2017-07-14 | 2017-09-19 | 长沙沁才生物科技有限公司 | Applications of the SPNP I in Nav1.3 passage tool reagents are prepared |
| CN107163127A (en) * | 2017-07-14 | 2017-09-15 | 长沙沁才生物科技有限公司 | Applications of the SPNP I in Nav1.4 passage tool reagents are prepared |
| CN107163126A (en) * | 2017-07-14 | 2017-09-15 | 长沙沁才生物科技有限公司 | Applications of the SPNP I in Nav1.7 passage tool reagents are prepared |
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