WO2018028117A1 - 一种蝎毒耐热合成肽及其用途 - Google Patents

一种蝎毒耐热合成肽及其用途 Download PDF

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WO2018028117A1
WO2018028117A1 PCT/CN2016/112078 CN2016112078W WO2018028117A1 WO 2018028117 A1 WO2018028117 A1 WO 2018028117A1 CN 2016112078 W CN2016112078 W CN 2016112078W WO 2018028117 A1 WO2018028117 A1 WO 2018028117A1
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scorpion
resistant
peptide
heat
synthetic peptide
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PCT/CN2016/112078
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French (fr)
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赵杰
李韶
张万琴
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大连医科大学
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Priority to CA2984569A priority Critical patent/CA2984569C/en
Priority to ES16905684T priority patent/ES2945407T3/es
Priority to JP2017554073A priority patent/JP6483858B2/ja
Priority to EP16905684.3A priority patent/EP3505530B1/en
Priority to US15/800,802 priority patent/US10442837B2/en
Publication of WO2018028117A1 publication Critical patent/WO2018028117A1/zh
Priority to US16/573,495 priority patent/US10870680B2/en

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Definitions

  • the invention belongs to the field of polypeptide drug research and development, and particularly relates to an amino acid sequence of a scorpion venom heat-resistant peptide (SVHRP) obtained from a traditional Chinese medicine scorpion venom and its synthetic product scorpion venom heat-resistant synthetic peptide (SVHRSP) in the treatment of epilepsy, Alz Application of Haimer's disease and Parkinson's disease.
  • SVHRP scorpion venom heat-resistant peptide
  • SVHRSP synthetic product scorpion venom heat-resistant synthetic peptide
  • PD Parkinson's Disease
  • DA dopamine
  • NCs pars compacta
  • BmK Buthus martensii Karsch
  • SmK Scorpion Venom
  • toxin action mechanism it can be divided into neurotoxin and cytotoxin.
  • neurotoxin is classified into long-chain scorpion toxin (containing 60-70 amino acid residues) and short-chain scorpion toxin (containing 30-40 amino acid residues).
  • the target of long-chain scorpion toxin is mainly voltage-dependent Na + channels on the neuro-excitable membrane, and short-chain scorpion toxin can act on Ca 2+ channel, K + channel or Cl - aisle.
  • Scorpion toxin has been widely used in the development of tools and antitoxins for membrane ion channels. It is a traditional Chinese medicine in China. So far, China has isolated and purified the scorpion toxin from the BmK scorpion venom with anti-tumor, anti-pain, anti-epileptic, anti-thromb, anti-inflammatory, anti-rheumatic and antibacterial functions. The toxicity of scorpion venom is second only to snake venom.
  • the national invention patent CN1324621 confirmed the effectiveness of the venom-scorpion venom-removed epilepsy (RE) on the medicinal part of the natural drug, and the safety of the Refractory Epilepsy (RE).
  • the National Invention Patent Application discloses the removal of toxic components which are not heat-resistant and heat-resistant from the SV of BmK, and obtains a safer extract of scorpion-resistant heat-resistant peptide.
  • the scorpion venous peptide extract has a low peptide yield, so obtaining chemically synthesized scorpion phytol peptide is a key link in determining whether this original research and development can be transformed and developed and industrialized.
  • the content of the application mainly includes the determination of the amino acid sequence of the scorpion phytolophilic peptide, the solid phase chemical synthesis according to the sequence, and the pharmacodynamic activity and safety detection of the scorpion venom heat-resistant synthetic peptide (SVHRSP).
  • the amino acid sequence of the scorpion venom resistant synthetic peptide (SVHRSP) of the present invention is as follows:
  • SEQ ID No. 1 (N-terminal) Lys-Val-Leu-Asn-Gly-Pro-Glu-Glu-Glu-Ala-Ala-Ala-Pro-Ala-Glu (C-terminal) scorpion venom heat-resistant synthetic peptide
  • a synthetic active polypeptide comprising 15 amino acid residues having a molecular weight of 1524 Da.
  • the scorpion venom heat-resistant synthetic peptide (SVHRSP) of the present invention first obtains the amino acid sequence of scorpion phytohormone from the traditional Chinese medicine BmK scorpion venom: mainly includes an experimental sample confirmed by an animal experiment (xiedu 20160112-peptide summary) for refractory epilepsy, Parkinson's disease and senile dementia have obvious preventive and therapeutic effects; after the sample is further subjected to LaGM composite material and repeated rapid magnetic separation, the mass spectrometry parallel experiment of nanoliter-electrospray ionization mass spectrometry (nanoLC-ESI-MS) is used for detection.
  • nanoLC-ESI-MS nanoliter-electrospray ionization mass spectrometry
  • scorpion phytohormone obtained by solid phase chemical synthesis, chromatographic purification and mass spectrometry to obtain a scorpion venom resistant synthetic peptide (SVHRSP) whose amino acid sequence is shown in SEQ ID No. 1, this sequence is maintained.
  • SVHRSP scorpion venom resistant synthetic peptide
  • the method for extracting the venom heat-resistant peptide of the present invention is as follows:
  • the sample was coarsely divided by Superdex Peptide 10/300GL molecular sieve column (Optimum Separation range (peptides) M, 100-7000 Da) (see Figure 1), and the sample was subdivided by HPLC (see Figure 2).
  • the chromatographic conditions were: :Zorbax SB-C18 4.6*250 5 ⁇ m (AgilentUSA), mobile phase: liquid A: acetonitrile/water: 2:98 (containing 0.1% trifluoroacetic acid); liquid B: acetonitrile/water: 98:2 (containing trifluoroacetic acid 0.08%); 0–40% B approximately 3-6 column volumes, 40–100% B 0.5-1 column volumes, 100% B 1-3 column volumes (CV); flow rate: 0.8 ml/min; UV The detector has a detection wavelength of 280 nm / 258 nm / 214 nm.
  • the polypeptide synthesis technique is to obtain a target molecule by orienting an amide bond method according to the amino acid sequence of the scorpion phytolophilic peptide.
  • Solid phase synthesis is to connect the carboxyl group of one amino acid of the drug substance to the solid phase carrier (Fmoc resin) in the form of a covalent bond, and then use the amino group of this amino acid as the starting point of synthesis to make the carboxyl group with the adjacent amino acid (amino group).
  • An acylation reaction occurs to form a peptide bond.
  • the amino group of the resin peptide containing these two amino acids is then deprotected and reacted with the carboxyl group of the next amino acid, and the process is repeated until the target peptide is formed.
  • the scorpion venom heat-resistant synthetic peptide of the present invention uses the Morris water maze test method to detect the effect of scorpion venom heat-resistant synthetic peptide (SVHRSP) on learning and memory of AD mice, and the results indicate that scorpion venom heat-resistant synthetic peptide (SVHRSP) has space for AD mice.
  • SVHRSP scorpion venom heat-resistant synthetic peptide
  • scorpion venom heat-resistant synthetic peptide SVHRSP
  • the toxic heat-resistant synthetic peptide SVHRSP
  • the effects of scorpion venom-resistant synthetic peptide (SVHRSP) on type II astrocytes were observed.
  • the results showed that scorpion venom-resistant synthetic peptide (SVHRSP) can promote type II astrocyte reprogramming (reverse differentiation) into brain. Endogenous neural stem cells.
  • SVHRSP scorpion venom-resistant synthetic peptide
  • Figure 1 is a gel filtration chromatogram of a sample of a venom heat-resistant peptide extract
  • Fig. 3 Morris water maze test to detect the effect of scorpion venom heat-resistant synthetic peptide on spatial learning and memory ability in senile dementia (AD) mice;
  • Figure 4 venom heat-resistant synthetic peptide scavenging 6-OHDA PD cell model ROS production
  • Figure 5 venom-resistant synthetic peptide inhibits epileptic discharge of hippocampal slices
  • Figure 7 shows the results of MS identification of venom-resistant synthetic peptide
  • Figure 10 Inhibition of sodium currents in primary cultured neurons by venom-resistant synthetic peptides
  • Figure 11 venom heat-resistant synthetic peptide has protective effect on NMDA-induced SH-SY5Y cell injury
  • Figure 12 venom heat-resistant synthetic peptide promotes type II astrocyte reprogramming (reverse differentiation) endogenous stem cells in the brain;
  • Figure 13 venom heat-resistant synthetic peptide promotes dedifferentiation of OPC cells into endogenous stem cells
  • Figure 14 The venom heat-resistant synthetic peptide up-regulates Nestin protein expression.
  • the centrifugal ultrafiltration tube with molecular weight cutoff of 50kDa and 30kDa filter is used to centrifuge the ultrafiltration of scorpion toxic heat-resistant component extract. Liquid, to obtain a venom heat-resistant peptide extract.
  • the muscarinic peptide extract was subjected to coarse fractionation using Superdex Peptide 10/300GL molecular sieve column (Optimum Separation range (peptides) M, 100-7000 Da) (see Figure 1), and the sample was subdivided by HPLC (see Figure 2).
  • the chromatographic conditions were: column: Zorbax SB-C18 4.6*250 5um (Agilent.USA), mobile phase: liquid A: acetonitrile/water: 2:98 (containing 0.1% trifluoroacetic acid); liquid B: acetonitrile/water : 98:2 (containing 0.08% trifluoroacetic acid); 0–40% B approximately 3-6 column volumes, 40–100% B 0.5-1 column volumes, 100% B 1-3 column volumes (CV) ; Flow rate: 0.8 ml/min; detection wavelength: 280 nm / 258 nm / 214 nm for subdivision. A purified extract of the venom heat-resistant polypeptide is obtained.
  • the analytical column is a C18 reversed phase column (75 ⁇ m x 15cm C18-3 ⁇ m) ChromXPEksigent), the gradient used in the experiment was increased from 5% to 80% in 70 min.
  • the scanning time of the _MS single image is 250ms. Under the IDA cycle, a maximum of 35 secondary spectra with 2+ to 8+ charge and a single second count greater than 100 are collected. The cumulative time of each secondary spectrum is 80ms. Each cycle time was fixed at 2.5 seconds and the collision cell energy setting was applied to all precursor ion collision induced dissociation (CID) with a dynamic exclusion setting of 11 seconds.
  • CID precursor ion collision induced dissociation
  • the scorpion venous heat-resistant peptide (SVHRP) extract was freeze-dried by LaGM composite and repeated rapid magnetic separation.
  • the lyophilized polypeptide sample was redissolved in Nano-RPLC Buffer A, the online Nano-RPLC liquid chromatography in EksigentnanoLC-Ultra TM 2D system (AB SCIEX), maintaining a flow rate of flushing desalted 10min.
  • Mass spectrometry was performed using a TripleTOF 5600 system (AB SCIEX) in combination with a nanoliter spray III ion source (AB SCIEX, USA), the original wiff map file acquired by mass spectrometry, and data processing using Protein Pilot Software v.4.5 (AB SCIEX, USA) software. And search analysis, the amino acid sequence of the scorpion venom polypeptide was detected.
  • Solution B containing 0.1% trifluoroacetic acid water
  • the water maze detects behavioral changes, and the navigational experiment lasts for 5 days, training 4 times a day, recording the time when the mouse finds the platform (the platform is placed in the first quadrant), that is, escaping the incubation period, and letting the mouse stay on the platform for 20s; if small No platform was found in the mouse for 60s, and the escape latency was recorded as 60s.
  • the space search experiment removed the platform after 4 trainings on the 5th day of the navigation test, and then placed the mice in the pool at the third quadrant water inlet to start the test.
  • the swimming distance and swimming time percentage of the mouse in the target quadrant (1st quadrant) were calculated for a total duration of 60 s. The number of mice crossing the hidden platform was measured.
  • the hidden platform of water maze obtained the escape latency of mice in the AD model group on the experimental test day (Day 5) (5d, 12 ⁇ 2) Time) was significantly shorter than the escape latency of the AD model group (day 5, 22 ⁇ 2 times) **p ⁇ 0.01, and the swimming distance was reduced from 500 cm on the 5th day of the model group to 300 cm in the g administration group.
  • the 5th increase in the model group was 8 times in the drug-administered group.
  • Synchronized transgenic nematode CL2355 and its control strain CL2122 were given different concentrations of SVHRSP (2, 20, 40 ⁇ g/ml) and the same amount of three distilled water, and were administered from the eggs, and the nematodes were transferred to new drugs every day.
  • the NGM dish After all the nematodes were cultured at 16 ° C for 36 hours, the incubator temperature was raised to 23 ° C, and further culture was carried out for 36 hours. The above nematodes were collected and washed 3 times with M9 buffer.
  • the petri dish used for the chemotaxis experiment was a 100 mm dish.
  • the solid medium was mixed with 1.9% agar, 1 mM CaCl 2 , 1 mM MgSO 4 , and 25 mM phosphate buffer, pH 6.0, and then poured into a dish and cooled. At the bottom of the Petri dish, two vertical lines are drawn with a marker on the center of the dish, and the whole dish is divided into four quadrants. About 60 nematodes in each group were placed in the center of the chemotaxis, and 1 ⁇ l of benzaldehyde (concentrated at 0.1%, diluted with absolute ethanol) and 1 ⁇ 0.25 M sodium azide were dropped to the center of the 1 and 3 quadrants.
  • Benzaldehyde is a chemical substance with a high concentration of odor. The nematode is attracted to the odor of benzaldehyde and moves to its area. When exposed to sodium azide, it will be paralyzed in situ.
  • the chemotaxis index is calculated by counting the number of nematodes in the 1 and 3 quadrants (the quadrant of the attraction) - the number of nematodes in the 2 and 4 quadrants divided by the total number of nematodes.
  • the scorpion venom-resistant synthetic peptide can increase the CI of CL2355 in a dose-dependent manner.
  • the protective effect of 40 ⁇ g/ml SVHRSP on neurons is most obvious, and the chemotaxis behavior damage caused by A ⁇ expression is almost completely reversed.
  • p ⁇ 0.01 compared with the two indicates that SVHRSP can protect neurons, counteract the toxic effects caused by A ⁇ , and improve the expression of A ⁇ caused by neurons.
  • the chemotaxis behavior is abnormal, see Table 2.
  • Fluorescence probe DCFH-DA was used to detect the effect of SVHRSP on 6-OHDA-induced SH-SY5Y oxidative stress.
  • the experiment was divided into normal control group, ROSup positive control group, 6-OHDA model group and different concentrations (2 ⁇ g/ml, 5 ⁇ g). /ml, 10 ⁇ g/ml, 20 ⁇ g/ml) SVHRSP drug group.
  • the SH-SY5H cells in the exponential growth phase were taken, the culture solution was discarded, washed once with PBS after sterilization, and then added with 0.25% trypsin for digestion and blown into individual cells.
  • the cells were counted by a cell counting plate, seeded in a 96-well plate at 1.5 ⁇ 10 4 /ml, and the cells were allowed to settle at the bottom of the plate, placed in a 5% CO 2 , and cultured in a 37 ° C incubator for 24 hours. After adding the concentration of 20 ⁇ g/ml SVHRSP drug group for 1 h, 6-OHDA was added, and 5% CO 2 was further added, and cultured in a 37 ° C incubator for 24 hours.
  • the ROSup was diluted with the medium at a dilution ratio of 1:500, 100 ⁇ l was added to each well, cultured for 27 minutes, and cultured for 27 minutes, diluted with DCFH-DA in a serum-free medium at a dilution ratio of 1:2000, and discarded at a final concentration of 5 ⁇ mol/L.
  • the medium was added with 50 ⁇ l of diluted DCFH-DA solution per well, gently shaken, placed in 5% CO 2 , and cultured in a 37 ° C incubator for 20 min. The supernatant was discarded, washed 3 times with PBS, and detected by fluorescence detection at 488 nm excitation wavelength and 525 nm emission wavelength in a fluorescent plate reader. Measure the OD value and compare.
  • SVHRSP for 6-OHDA-induced SH-SY5Y intracellular production
  • 6-OHDA 100 ⁇ M was given for 24h.
  • the expression of ROS in the cells was detected by fluorescence microplate reader at excitation wavelength of 488nm and emission wavelength of 525nm.
  • 6-OHDA can significantly increase intracellular ROS and is statistically significant (control 1 ⁇ 0.2vs6-OHDA 3.4 ⁇ 0.4p ⁇ 0.01), but when added to SVHRSP, it can clear intracellular ROS, and also Statistically significant (control 3.4 ⁇ 0.4 vs 1.5 ⁇ 0.4 p ⁇ 0.05).
  • ip pilocarpine epilepsy model + NS
  • lithium-pilocarpine epilepsy model + sodium valproate Observe and record the level of epilepsy, according to Racine , s classification as the evaluation criteria:
  • SVHRSP has a significant control effect on seizures in chronic model of rats with lithium-pilocarpine epilepsy, which can significantly reduce the number of seizures;
  • SVHRSP is stronger than the positive control valproic acid in reducing the level of seizure and reducing the number of seizures in the chronic model of rats with lithium-pilocarpine epilepsy.
  • the ml ml-coated plates were placed in an incubator at 37 ° C and 10% CO 2 for cultivation.
  • all the planting liquids were replaced with the culture solution, and on the 4th day, cytarabine (3 ⁇ g/ml) was added to the culture solution to inhibit excessive proliferation of non-neuronal cells.
  • 50% of the fresh culture medium was replaced every 3 days, and the hippocampal neurons cultured in vitro were cultured until 9-12 days, and the neurons were cultured for 10 days.
  • the drugs used were all dosed through a BPS-8 perfusion device.
  • the diameter of each tube was 0.2 mm, and the tube was about 100 ⁇ m from the recorded cells.
  • the sodium channel current was recorded by patch clamp in the primary cultured neuron system, and the currents were compared before and after the same cells were added with scorpion refractory synthetic peptide (SVHRSP).
  • SVHRSP scorpion refractory synthetic peptide
  • SH-SY5Y cells are derived from human neuroblastoma cell lines.
  • NMDA model NMDA 20 mM + Gly 10 ⁇ M.
  • Experimental procedure Before the experiment, grouped according to the purpose of the experiment. SH-SY5Y was seeded in 96-well plates (0.8 ⁇ 10 4 /well). After 24 h, SVHRSP and peptide were added for pre-incubation for 24 h. Then, according to the group, NMDA 20 mM+Gly10 ⁇ M was added for 24 h, and the cell viability was detected by MTT kit. See Figure 11. The results showed that SVHRSP had protective effects on NMDA-induced SH-SY5Y cell injury.
  • Type II astrocytes form pluripotent neural stem cells with proliferation and differentiation ability by reverse differentiation
  • type II astrocyte inducing solution (20% DMEM). After 4-5 days, continue to culture. Relatively purified type II astrocytes can be obtained. After 5 days, type II astrocytes were digested and planted in ultra-low adhesion 6-well plates. After changing the culture medium for stem cell culture for 12 days, stem cell spheres were observed. After 12 days, the cells in the ultra-low adhesion 6-well plate were aspirated and placed in a 24-well plate covered with polylysine-treated cells. After 24 hours, the slides were taken out and fixed with 4% paraformaldehyde.
  • SVHRSP scorpion venom thermosuppressant peptide
  • MTD 1Maximal tolerancedose
  • MLD 2 Minimal lethal dose
  • the maximum non-toxic reaction dose (the highest non-toxic): the No observed adverse effect level (NOAEL), that is, the highest dose of no damage found in the animal within a certain period of time;
  • NOAEL No observed adverse effect level
  • the minimum dose of toxic reaction in an animal the dose that just caused the toxic reaction is the lowest dose at which the animal develops a toxic reaction.
  • Test animals Kunming mice, female and male (half healthy adult, weight 19-20g;
  • Test substance SVHRSP: a.
  • Source Henan Yichang Xinxin Yangshuo Plant (2012-05-28) purchased BmK scorpion venom extract;
  • the optimal administration capacity of the species of animals was selected according to the route of administration (different doses, but the administration volume was the same), and the ip administration route of this experiment was 0.2 ml/20 g BW.
  • MTD maximum tolerance dose
  • the range between the dose and the minimum lethal dose is a range of approximate lethal doses; if the animal dies at the dose, the dose is The range between the maximum tolerated doses is approximately the lethal dose range.
  • the results showed that the medicinal safety and pharmacodynamic activity ratio of the scorpion venom heat-resistant synthetic peptide (SVHRSP) of the present invention was greater than 2000/0.05 40,000 times.
  • the maximum non-toxic reaction dose (highest non-toxic) has not been reached: the maximum dose without toxicity.

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Abstract

本发明公开了一种蝎毒耐热合成肽及其用途。本发明的多肽为从东亚钳蝎(BmK)蝎毒中检出蝎毒耐热肽(SVHRP)的氨基酸序列，具有防治难治性癫痫、帕金森病和阿尔兹海默病(早老年痴呆)的药效活性，其氨基酸序列如SEQ ID No：1所示。

Description

一种蝎毒耐热合成肽及其用途 技术领域
本发明属于多肽药物研发领域，特别涉及一种中药东亚钳蝎蝎毒中获取蝎毒耐热肽(SVHRP)的氨基酸序列及其合成产品蝎毒耐热合成肽(SVHRSP)在治疗癫痫、阿尔茨海默病和帕金森病方面的应用。
背景技术
帕金森病(Parkinson’s Disease,PD)的临床症状的出现是由于中脑黑质内多巴胺(DA)神经元损伤导致纹状体DA神经递质水平的严重降低所致。PD氧化应激学说的提出，是由于DA氧化应激；自由基学说是中脑黑质致密部(NCs)DA神经元损伤的重要机制,抗氧化治疗是目前公认的有效的治疗方案。
中药东亚钳蝎(ButhusmartensiiKarsch,BmK)，BmK亦称之为马氏钳蝎,现代医学研究证明由蝎尾毒囊腺排放的蝎毒(Scorpion Venom,SV)富含毒素。按其毒素作用机理可分为神经毒素和细胞毒素。其神经毒素被划分为长链蝎毒素(含60-70个氨基酸残基)和短链蝎毒素(含30-40个氨基酸残基)。长链蝎毒素作用的靶位主要是在神经可兴奋膜上的电压依赖性钠离子通道(Voltage-dependent Na+channels)，短链蝎毒素能作用于Ca2+通道、K+通道或Cl-通道。蝎毒素己被广泛用于膜离子通道的工具药和抗毒素的研制。全蝎是我国传统中药,迄今,我国已从BmK蝎毒中分离纯化出具有抗肿瘤、抗痛、抗癲痫、抗栓、抗炎、抗风湿、抗菌等功能的蝎毒素。蝎毒的毒性仅次于蛇毒。国家发明专利CN1324621证实了天然药物全蝎的药用部位蝎尾节毒囊腺排放的毒液-蝎毒对难治性癫痫(Refractory Epilepsy,RE)的有效性及经工艺处理后的安全性。国家发明专利申请(申请号:201310330290.1)公开了从BmK的SV中,去除不耐热和耐热的有毒成分,获取一种更安全的蝎毒耐热肽提取液。该多肽用于防治难治性癫痫(Refractory Epilepsy,RE)、帕金森病(Parkinsons Disease,PD)、阿尔兹海默病(Alzheimer,s Disease,AD)具有共同的作用靶点和各自的特殊药效。可是蝎毒耐热肽提取液获肽率低，因此获取化学合成的蝎毒耐热肽是决定这一原创性研发成果能否进行转化研发和产业化生产的关键环节。
发明内容
本发明的目的是提供一种具有治疗化学合成的蝎毒耐热肽及其用途。本申请内容主要包括测定蝎毒耐热肽的氨基酸序列、按此序列进行固相化学合成和对蝎毒耐热合成肽(SVHRSP)的药效活性、安全性检测。
本发明的蝎毒耐热合成肽(SVHRSP)的氨基酸序列如下:
SEQ ID No.1：(N端)Lys-Val-Leu-Asn-Gly-Pro-Glu-Glu-Glu-Ala-Ala-Ala-Pro-Ala-Glu(C端)蝎毒耐热合成肽为人工合成的活性多肽，包含15个氨基酸残基，分子量为1524Da。
本发明的蝎毒耐热合成肽(SVHRSP)先从传统中药BmK蝎毒中获取蝎毒耐热肽的氨基酸序列:主要包括经动物实验证实样品(xiedu 20160112-peptide summary)对难治性癫痫、帕金森病、老年性痴呆有明显防治作用；样品再经LaGM复合材料和反复快速磁分离后,进行纳升反相色谱-电喷雾质谱(nanoLC-ESI-MS)连用的质谱平行实验，检出蝎毒耐热肽的氨基酸序列；最后经固相化学合成、色谱纯化和质谱鉴定获取一种蝎毒耐热合成肽(SVHRSP),其氨基酸序列如SEQ ID No 1所示，这一序列保持了蝎毒耐热肽的药效活性和安全性以及多种生物活性。
本发明的蝎毒耐热肽提取方法如下：
1.蝎毒耐热肽的氨基酸序列测定
(1)将BmK蝎毒的冷冻干粉复溶、离心，取上清液，100℃隔水加热，取出，离心取上清液，即为蝎毒耐热组分提取液，先后用截留分子量为50kDa和30kDa滤膜的离心超滤管，对蝎毒耐热组分提取液进行离心超滤，取上槽液，得到蝎毒耐热肽提取液。用Superdex Peptide10/300GL分子筛柱子(Optimum Separation range(peptides)M，100-7000Da)对样品进行粗分(见图1)，用HPLC对样品进行细分(见图2)，色谱条件为，色谱柱：Zorbax SB-C18 4.6*250 5μm(AgilentUSA)，流动相：A液：乙腈/水：2:98(含三氟乙酸0.1％)；B液：乙腈/水：98:2(含三氟乙酸0.08％)；0–40％B大约3-6个柱体积，40–100％B 0.5-1个柱体积，100％B 1-3个柱体积(CV)；流速：0.8ml/min；紫外检测器，检测波长为：280nm/258nm/214nm。
(2)冻干的多肽样品重新溶解于Nano-RPLC Buffer A中，在线Nano-RPLC液相色谱在EksigentnanoLC-UltraTM2D系统(AB SCIEX)进行，溶解后的样品以2μL/min的流速上样到C18预柱上(100μm×3cm，C18,3μm,
Figure PCTCN2016112078-appb-000001
)，保持流速冲洗脱盐10min。质谱采用TripleTOF5600系统(AB SCIEX)结合纳升喷雾III离子源(AB SCIEX,USA)，质谱采集到的原始wiff图谱文件，采用Protein Pilot Software v.4.5(AB SCIEX,USA)软件进行数据加工处理，得到蝎毒耐热合成肽(SVHRSP)见序列表SEQ ID No.1。
2.用固相化学合成方法制备蝎毒耐热合成肽
(1)多肽合成技术是按照蝎毒耐热肽的氨基酸顺序，通过定向形成酰胺键方法得到目标分子。固相合成是将原料药一个氨基酸的羧基以共价键的形式与固相载体(Fmoc树脂)相连,再以这一氨基酸的氨基为合成起点，使其与相邻氨基酸(氨基保护)的羧基发生酰化反应，形成肽键。然后让包含有这两个氨基酸的树脂肽的氨基脱保护后与下一个氨基酸的羧基反应，不断重复这一过程,直至目标肽形成为止。
Figure PCTCN2016112078-appb-000002
(2)高效液相色谱(HPLC)纯化在多肽合成过程中会产生一些与目标肽结构类似的杂肽(见图6)，例如因氨基酸消旋化产生的非对映异构体、因部分氨基酸未连接上产生的缺失 肽、因肽键断裂产生的断裂肽等，色谱条件如下：色谱柱：Inertsil ODS-SP 4.6mm*250mm，0.1％三氟乙酸乙腈-0.1％三氟乙酸水，紫外检测器，检测波长为214nm；
(3)用质谱技术对合成多肽进行结构确认，检索分析，检出蝎毒耐热合成肽(SVHRSP)的氨基酸序列见序列表SEQ ID No.1。
本发明的蝎毒耐热合成肽采用Morris水迷宫实验法检测蝎毒耐热合成肽(SVHRSP)对AD小鼠学习记忆的影响，结果表明蝎毒耐热合成肽(SVHRSP)对AD小鼠空间学习记忆能力具有促进作用；采用线虫趋化行为学实验方法检测Aβ神经毒性，结果表明蝎毒耐热合成肽(SVHRSP)能够保护神经元，对抗Aβ引起的毒性作用，改善由于神经元Aβ表达引起的趋化行为异常；观察了蝎毒耐热合成肽(SVHRSP)对难治性癲痫匹罗卡品模型大鼠的防治作用，实验结果表明蝎毒耐热合成肽(SVHRSP)对锂-匹罗卡品癫痫大鼠慢性模型痫性发作有显著的控制效果,可显著减少发作次数；观察了蝎毒耐热合成肽(SVHRSP)对原代培养神经元钠电流的抑制作用，实验结果表明蝎毒耐热合成肽(SVHRSP)能明显抑制原代培养的海马神经元体钠通道电流；对NMDA诱导的SH-SY5Y细胞损伤具有保护作用。观察了蝎毒耐热合成肽(SVHRSP)对II型星形胶质细胞的影响，结果表明蝎毒耐热合成肽(SVHRSP)能够促进II型星形胶质细胞重编程(逆分化)为脑内内源性神经干细胞。通过测细胞内ROS的方法来检测SVHRSP对6-OHDA导致的SH-SY5Y细胞内产生的活性氧的清除的能力，结果显示，SVHRSP，能够明显清除细胞内ROS，具有抑制PD氧化应激的作用。蝎毒耐热合成肽(SVHRSP)的药用安全性与药活性比大于2000/0.05＝40,000倍，昆明种小鼠腹腔注射途径给药剂量高至2000mg/kgBW仍末见任何毒性反应，显示出作为新型抗癫痫、阿尔茨海默病和帕金森病药物的应用前景。
附图说明
图1蝎毒耐热肽提取液样品的凝胶过滤层析色谱图；
图2蝎毒耐热肽提取液样品RP-HPLC色谱图；
图3Morris水迷宫实验检测蝎毒耐热合成肽对老年性痴呆(AD)小鼠空间学习记忆能力的影响；
图4蝎毒耐热合成肽清除6-OHDA PD细胞模型ROS产生；
图5蝎毒耐热合成肽抑制海马脑片癫痫样放电；
图6蝎毒耐热合成肽的反相-高效液相色谱(RP-HPLC)结果；
图7蝎毒耐热合成肽的MS鉴定结果；
图8蝎毒耐热合成肽对锂-匹罗卡品癫痫大鼠反复发作的抑制作用；
图9蝎毒耐热合成肽对AD的转基因秀丽线虫CL2355趋化性行为的影响；
图10蝎毒耐热合成肽对原代培养神经元钠电流的抑制作用；
图11蝎毒耐热合成肽对NMDA诱导的SH-SY5Y细胞损伤具有保护作用；
图12蝎毒耐热合成肽促进II型星形胶质细胞重编程(逆分化)脑内内源性干细胞；
图13蝎毒耐热合成肽促进OPC细胞去分化为内源性干细胞；
图14蝎毒耐热合成肽上调Nestin蛋白表达。
具体实施方式
以下实施例子进一步解释本发明的内容，但并不用以限制本发明。
实施例1
蝎毒耐热肽的氨基酸序列测定
(1)用三蒸水将由河南宜昌的BmK蝎毒的冷冻干粉复溶、离心,取上清液即BmK蝎毒提取液；用带盖耐热的塑料管分装后,置于恒温水浴槽中，100℃隔水加热4h后取出,自然降温至室温后离心、高速离心取上清液，即为蝎毒耐热组分提取液；先后用具有截留分子量为50kDa和30kDa滤膜的离心超滤管，对蝎毒耐热组分提取液进行离心超滤，取上槽液，得到蝎毒耐热多肽提取液。离心取上清液，即为蝎毒耐热组分提取液，先后用截留分子量为50kDa和30kDa滤膜的离心超滤管，对蝎毒耐热组分提取液进行离心超滤，取上槽液，得到蝎毒耐热肽提取液。
蝎毒耐热肽提取液用Superdex Peptide 10/300GL分子筛柱子(Optimum Separation range(peptides)M,100-7000Da)进行粗分(见图1)，用HPLC对样品进行细分(见图2)，色谱条件为，色谱柱：Zorbax SB-C18 4.6*250 5um(Agilent.USA)，，流动相：A液：乙腈/水：2:98(含三氟乙酸0.1％)；B液：乙腈/水：98:2(含三氟乙酸0.08％)；0–40％B大约3-6个柱体积，40–100％B 0.5-1个柱体积，100％B 1-3个柱体积(CV)；流速：0.8ml/min；检测波长为：280nm/258nm/214nm进行细分。得到蝎毒耐热多肽提取纯化物。
(2)取1.5ml蝎毒耐热多肽提取纯化物，10000rpm离心10min后取上清液；按照上清液:30mg/mL-LaGM复合材料＝50：1的比例,将30mg/mL-LaGM复合材料加入上清液，充分涡旋2min后1000r室温振荡10min；10000r离心5min磁分离，去掉上清，在沉淀中加入500ul水，充分涡旋1min，室温振荡5min，10000r离心5min磁分离，去掉上清，重复2次。在沉淀中加入20ul 80％+1％TFA的洗脱液，涡旋1min，室温振荡5min，10000r离心5min，磁分离，收集上清，重复1次；冷冻干燥。将磁性材料分离后冻干的多肽样品重新溶解于Nano-RPLC Buffer A中，在线Nano‐RPLC液相色谱在EksigentnanoLC‐UltraTM2D系统(AB SCIEX)进行，溶解后的样品以2μL/min的流速上样到C18预柱上(100μm×3cm，C18,3μm,
Figure PCTCN2016112078-appb-000003
)，然后保持流速冲洗脱盐10min。分析柱是C18反相色谱柱(75μm x 15cm C18-3 μm
Figure PCTCN2016112078-appb-000004
ChromXPEksigent)，实验所用梯度为70min内流动相B由5％升高至80％。质谱采用Triple TOF 5600系统(AB SCIEX)结合纳升喷雾III离子源(AB SCIEX,USA)，喷雾电压为2.4kV，气帘气压为30Psi，雾化气压为5Psi，加热温度为150℃，一级TOF‐MS单张图谱扫描时间为250ms，每次IDA循环下最多采集35个电荷为2+到8+且单秒计数大于100的二级图谱，每张二级图谱的累积时间为80ms。每次循环时间固定为2.5秒，碰撞室能量设定适用于所有前体离子碰撞诱导解离(CID)，动态排除设置为11秒。
(3)数据分析条件
质谱采集到的原始wiff图谱文件，采用Protein Pilot Software v.4.5(AB SCIEX,USA)软件进行数据加工处理和检索分析，数据库为uniprot库中的蝎子，检索方式为彻底分析，假阳性率控制为1％FDR。得到蝎毒耐热合成肽(SVHRSP)，其氨基酸序列见序列表SEQ ID No.1；
检索结果如表1.
表1.nanoLC-ESI-MS连用的质谱平行实验,检出蝎毒耐热肽的氨基酸序列
Figure PCTCN2016112078-appb-000005
样品Lot No:xiedu 20160112-peptide summary.Txt Microsoft Excel
蝎毒耐热肽(SVHRP)提取液经LaGM复合材料和反复快速磁分离后，冷冻干燥。冻干的多肽样品重新溶解于Nano-RPLC Buffer A中，在线Nano-RPLC液相色谱在EksigentnanoLC-UltraTM2D系统(AB SCIEX)进行，保持流速冲洗脱盐10min。质谱采用TripleTOF 5600系统(AB SCIEX)结合纳升喷雾III离子源(AB SCIEX,USA)，质谱采集到的原始wiff图谱文件，采用Protein Pilot Software v.4.5(AB SCIEX,USA)软件进行数据加工处理和检索分析，检出蝎毒耐热多肽的氨基酸序列。
实施例2
合成蝎毒耐热多肽高效液相色谱(HPLC)纯化
在多肽合成过程中会产生一些与目标肽结构类似的杂肽，例如因氨基酸消旋化产生的非对映异构体、因部分氨基酸未连接上产生的缺失肽、因肽键断裂产生的断裂肽等。因此用 RP-HPLC进行纯化:
样品纯化条件:
结构:KE-15
序号:0200046
批号:P160418-CQ455216
柱子:4.6mm*250mm,Inertsil ODS-SP
溶液A:含0.1％三氟乙酸乙腈
溶液B:含0.1％三氟乙酸水
Figure PCTCN2016112078-appb-000006
流速:1.0ml/min
波长:214nm
进样体积:10ìl
实施例3
用Morris水迷宫实验检测SVHRSP对AD小鼠学习记忆的影响
水迷宫检测行为学改变,定位航行实验历时5d，每天训练4次，记录小鼠找到平台(平台放置在第1象限)的时间，即逃避潜伏期，并让小鼠在平台上停留20s；如果小鼠60s内未找到平台，逃避潜伏期记为60s。空间搜索实验在定位航行试验第5d的4次训练之后，撤除平台，然后在第3象限入水点将小鼠放入池中，开始测试。计算小鼠在目标象限(第1象限)内游泳距离和游泳时间百分比，总时长60s。检测小鼠穿越隐匿平台次数。
Morris水迷宫实验研究发现SVHRSP对AD小鼠空间学习记忆能力具有促进作用，水迷宫的隐藏平台获得实验测试日(第5天)AD模型给药组小鼠的逃避潜伏期(第5d，12±2次)明显短于AD模型组(第5天，22±2次)的逃避潜伏期**p<0.01,其游泳距离由模型组的第5d的500cm降低为g给药组300cm，通过平台次由模型组的5次上升为给药组8次。
实施例4
线虫趋化行为学实验方法
同期化的转基因线虫CL2355及其对照株CL2122分别给予不同浓度SVHRSP(2，20，40μg/ml)及等量的三蒸水，从虫卵时开始给药，每天将线虫转移到新的含有药物的NGM培养皿中。所有的线虫在16℃培养36小时后，升高培养箱温度到23℃，再进行36小时的培养。将以上线虫收集起来，用M9缓冲液清洗3次。进行趋化实验所用培养皿用100mm大皿。固体培养基含1.9％琼脂，1mM CaCl2，1mM MgSO4，and 25mM磷酸盐缓冲液，pH 6.0微波 混匀后，倒入皿中，冷却即可。在培养皿底部经过中心位置用马克笔画两条互相垂直的直线，将整个皿均分为四个象限。每组约60条线虫放到趋化培养皿的中心位置，将1μl苯甲醛(浓度为0.1％，无水乙醇稀释)和1μ0.25M叠氮钠滴到1、3象限的中心位置，1μl无水乙醇和1μl 0.25M叠氮钠滴到2、4象限，该点距离中心点的距离应大于2厘米。将趋化培养皿转移到23℃培养箱中，一个小时后，计数趋化指数。苯甲醛是一种高浓度气味的化学物质，线虫被苯甲醛的气味吸引会向其区域移动，接触到叠氮钠就会被麻痹在原地。趋化指数的计算方法为计数在1、3象限(吸引物所在象限)的线虫数量-在2、4象限线虫数量再除以线虫总数量。
蝎毒耐热合成肽(SVHRSP)能够剂量依赖性的提高CL2355的CI，40μg/ml的SVHRSP对神经元的保护作用最为明显，几乎完全逆转了Aβ表达所致的趋化性行为损伤。由对照组(39.45±6.2)的变为给药组(7±5.66)，二者相比p<0.01这表明SVHRSP能够保护神经元，对抗Aβ引起的毒性作用，改善由于神经元Aβ表达引起的趋化行为异常，见表2。
表2线虫趋化行为学结果
Figure PCTCN2016112078-appb-000007
实施例5
SVHRSP对6-OHDA致SH-SY5Y氧化应激的影响
荧光探针DCFH-DA检测ROS观察SVHRSP对6-OHDA致SH-SY5Y氧化应激的影响，实验分为正常对照组，ROSup阳性对照组，6-OHDA模型组及不同浓度(2μg/ml，5μg/ml，10μg/ml，20μg/ml)SVHRSP药物组。取处于指数生长期的SH-SY5H细胞，弃去培养液，用灭菌后PBS洗一次，再加入0.25％胰酶进行消化，吹打成单个细胞。用细胞计数板进行细胞计数，以1.5×104个/ml接种于96孔板，待细胞沉于板底，放到5％CO2,37℃培养箱中进行培养24h。加入浓度为20μg/ml SVHRSP药物组进行干预1h后，然后加入6-OHDA，继续放入5％CO2,37℃培养箱中进行培养24h。按照1：500的稀释比例用培养基稀释ROSup，每孔加入100μl，培养27分钟培养27min后，按照1:2000稀释比例用无血清培养基稀释DCFH-DA，使终浓度为5μmol/L弃去培养基，每孔加入50μl稀释好DCFH-DA溶液，轻轻摇匀，放入5％CO2，37℃培养箱中培养20min。弃去上清，用PBS洗3次，在荧光酶标仪中测用488nm激发波长，525nm发射波长进行荧光检测。测OD值，进行比较。
通过测细胞内ROS的方法来检测SVHRSP对6-OHDA导致的SH-SY5Y细胞内产生 的活性氧的清除的能力。给予SVHRSP(20ìg/ml)预先处理1h，再给予6-OHDA(100ìM)损伤24h，利用荧光酶标仪，在激发波长为488nm，发射波长为525nm，检测细胞内ROS的表达情况，实验结果显示，6-OHDA能使细胞内的ROS明显升高，并且具有统计学意义(control 1±0.2vs6-OHDA 3.4±0.4p<0.01)，但是当加入SVHRSP时，能够明显清除细胞内ROS，而且也具有统计学意义(control 3.4±0.4vs 1.5±0.4p<0.05)。
实施例6
蝎毒耐热合成肽(SVHRSP)对难治性癲痫匹罗卡品模型大鼠的防治作用
1)动物选择与分组选取体重为180g健康雄性SD大鼠,锂-匹罗卡品ip 300mg/kg诱发颞叶癫痫发作；癫痫急性发作后15天,将癫痫大鼠随机分为模型组和模型给药组；癲痫模型组动物被给予腹腔注射生理盐水(NS),连续15天；癫痫模型给药组大鼠又被分成3个模型给药组,分别给予SVHRP提取液50ug/kg/d、ip；SVHRSP 50ug/kg/d、ip和阳性对照药物-丙戊酸钠：即匹罗卡品癫痫模型+NS；锂-匹罗卡品癫痫模型+SVHRP；锂-匹罗卡品癫痫模型+SVHRSP；锂-匹罗卡品癫痫模型+丙戊酸钠。观察并记录癫痫级别,按Racine,s分级作为评价标准:
表3.癫痫发作级别
Figure PCTCN2016112078-appb-000008
结果分析:
(1)SVHRSP对锂-匹罗卡品癫痫大鼠慢性模型痫性发作有显著的控制效果,可显著减少发作次数；
(2)SVHRSP在降低锂-匹罗卡品癫痫大鼠慢性模型的发作级别与减少发作次数上均强于阳性对照组丙戊酸
Figure PCTCN2016112078-appb-000009
Figure PCTCN2016112078-appb-000010
实施例7
蝎毒耐热合成肽(SVHRSP)对原代培养神经元钠电流的抑制作用
(1)原代培养的海马神经元培养。出生24h SD大鼠用75％酒精灭菌，冰上断头，解剖显微镜下快速完整剥离双侧海马，于解剖液中分成体积为1mm3小块，放入终浓度为0.125％的胰酶消化液中，置于37℃、10％CO2孵箱中消化30min。取出消化好的组织，在种植液中终止消化，机械吹打、悬浮、筛网过滤，制成密度为1×105/ml的细胞悬液，接种于事先用多聚赖氨酸(0.1mg/ml)包被的培养板中，放入37℃、10％CO2的培养箱内进行培养。第2d将种植液全部换为培养液，第4d在培养液中加入阿糖胞苷(3μg/ml)，抑制非神经细胞的过度增殖。以后每隔3d更换50％新鲜培养液，体外培养的海马神经元在培养到第9-12d为成熟神经元，本实验采用培养10d以后的神经元。
(2)全细胞膜片钳记录原代培养的海马神经元培养10d后进行全细胞膜片钳记录。记录玻璃微电极外径1.5mm，内径0.6-0.8mm，长8cm，用PP-830型微电极拉制仪经双步垂直拉制而成。拉制后电极口径约为1μm，充灌电极内液后，电极阻抗为3-5MΩ。参考电极为Ag-AgCl,电极。全细胞膜片钳记录电极与细胞膜之间形成稳定的高阻封接后，进行快电容补偿(C-fast)，稍加负压破膜，形成全细胞构型，再进行慢电容补偿(C-slow)，慢电容补偿率为80％-85％。通过漏电流法减去漏电流(Leak Substraction)。在设定的刺激电压下观察电流的激活情况。全细胞膜片钳记录所用仪器的设置为EPC-10双通道膜片钳放大器实验参数、数据的采集和刺激的施加均通过Pulse软件来控制，Bessel滤波1的频率10KHz，Bessel滤波2的频率2.9KHz。实验过程中，所用药物都通过BPS-8灌流装置进行加药，排管每管直径为0.2mm，管口距所记录细胞大约100μm。在原代培养的神经元体系进行膜片钳记录钠通道电流，对同一细胞加蝎毒耐热合成肽(SVHRSP)前后电流进行比较。
全细胞膜片钳记录，对同一细胞加蝎毒耐热合成肽(SVHRSP)前后电流进行比较，结果发现2μg/ml的SVHRSP能明显抑制原代培养的海马神经元体钠通道电流，抑制率达90％以上(n＝6)(p<0.0001)，给予细胞外液冲洗后电流可恢复。
实施例8
SH-SY5Y细胞衍生于人的神经母细胞瘤细胞系。NMDA模型：NMDA 20mM+Gly10μM.实验流程：实验前，按实验目的分组。SH-SY5Y种于96孔板(0.8×104/孔)，24h后加入SVHRSP和peptide预孵育24h，然后根据分组加入NMDA 20mM+Gly10μM培养24h后，用MTT试剂盒检测各种细胞存活率，见图11。结果表明，SVHRSP对NMDA诱导的SH-SY5Y细胞损伤具有保护作用。其中，SVHRP浓度为：2μg/ml，20μg/ml；SVHRSP浓度为：2μg/ml，20μg/ml；NMDA:20mM+Gly10μM，p<0.05(n＝3)。
实施例9
II型星形胶质细胞通过逆分化形成具有增殖及分化能力的多潜能神经干细胞
实验操作:新生24小时SD大鼠，无菌分离出大脑皮层，制成细胞悬液，在含10％胎牛血清的DMEM培养液中进行混合胶质细胞的原代培养。培养7-9d后，首先用L-亮氨酸甲酯盐酸盐(L-leucine methyl ester hydrochloride)处理1h，更换新鲜无菌的细胞培养液，于37℃恒温摇床180r/min震荡18小时后取上清液经200目筛网过滤，将上清液植于经多聚赖氨酸处理过的6孔中获得相对纯化的OPC。让细胞在孵箱中自然沉降，2h后观察细胞是否贴壁，贴壁后将上清液弃掉更换成II型星形胶质细胞诱导液(20％DMEM)，继续培养4-5d后，即可获得相对纯化的II型星形胶质细胞。5d后，将II型星形胶质细胞消化下来植于超低粘附6孔板中，更换培养液为干细胞培养液继续培养12d后，可见干细胞球。12d后将超低粘附6孔板中细胞球吸出植于铺有经多聚赖氨酸处理细胞爬片的24孔板中，24h后将爬片取出，用4％多聚甲醛固定，行免疫荧光染色，用Nestin(green)鉴定细胞球是否为干细胞。Nestin是神经干细胞的特异性标记物蝎毒耐热合成肽(SVHRSP)影响II型星形胶质细胞逆分化形成神经干细胞标志物Nestin。
实例10
蝎毒耐热合成肽(SVHRSP)的急性毒性试验
1)主要观察指标:
①最大耐受量(Maximal tolerancedose,MTD,即最高非致死剂量,指不引起受试动物死亡的最高剂量；
②最小致死剂量(Minimal lethal dose,MLD)：引起个别受试动物出现死亡的剂量；
③最大无毒性反应剂量(最高无毒):即未见毒性反应的最高剂量(No observed adverse effect level,NOAEL),即动物在一定时间内,未发现损害作用的最高剂量；
④最小毒性反应剂量：动物出现毒性反应的最小剂量:刚刚引起毒性反应的剂量即动物出现毒性反应的最低剂量。
2)注明:
①受试动物:昆明种小鼠雌、雄各半(健康成年,体重19-20g；
②受试物:SVHRSP:a.来源:河南宜昌鑫鑫养蝎厂(2012-05-28)购入BmK蝎毒提取液；
b.Lot No:P160418-CQ455216；
c.含量(或规格):10mg/支；d.保存条件及配制方法:长期保存-20℃,7-10d保存在4℃,使用时用millipore water复溶。
③给药途径:分别为ip(小鼠ip途径给药常用-最大容积为0.1-1.0ml/20gBW)
按给药途径选择该种属动物的最佳给药容量(剂量不同,但给药容量相同),本次实验ip给药途径均采用0.2ml/20gBW。
④选择采用方法
按近似致死剂量法,列出可能序列表:
按照从前蝎毒耐热多肽对昆明种小鼠的急性毒性试验结果,估计出蝎毒耐热合成肽(SVHRSP)可能的最大耐受剂量和最小致死剂量,然后按50％递增法，设计出含数个剂量的剂量序列表:
可能的最大耐受剂量(Maximal tolerance dose,MTD)：76.8、51.2、34.1mg/kg
可能的最小致死剂量(Minimal lethal dose,MLD)：115.2、76.8、51.2mg/kg
由估计的剂量序列表中找出可能的致死剂量范围(115.2、76.8、51.2、34.1mg/kg，在此范围内，每一个剂量给一只动物，测出最小致死剂量和最大耐受剂量，然后用二者之间的剂量给一只动物。如果该剂量下动物未发生死亡，则该剂量与最小致死剂量之间的范围为近似致死剂量范围；如果该剂量下动物死亡，则该剂量与最大耐受剂量间的范围为近似致死剂量范围。
表4.腹途径给药检测蝎毒耐热合成肽的安全性(最大耐受和最小致死剂量)
Figure PCTCN2016112078-appb-000011
Figure PCTCN2016112078-appb-000012
结果表明:本发明的蝎毒耐热合成肽(SVHRSP)的药用安全性与药效活性比大于2000/0.05＝40,000倍昆明种小鼠腹腔注射途径给药剂量高至2000mg/kg.BW仍末见任何毒性反应。尚未达到最大无毒性反应剂量(最高无毒):即未见毒性反应的最大剂量。

Claims (3)

  1. 一种蝎毒耐热合成肽，其特征在于：基因全序列见序列表SEQ ID No.1。
  2. 一种蝎毒耐热合成肽制备方法，其特征在于：
    (1)蝎毒耐热肽的氨基酸序列测定
    1)将BmK蝎毒的冷冻干粉复溶、离心，取上清液，、100℃隔水加热，取出，离心取上清液，即为蝎毒耐热组分提取液，先后用截留分子量为50kDa和30kDa滤膜的离心超滤管，对蝎毒耐热组分提取液进行离心超滤，取上槽液，得到蝎毒耐热肽提取液(见附图1、2)。用Superdex Peptide 10/300GL分子筛柱子(Optimum Separation range(peptides)M,100-7000Da)对样品进行粗分，用HPLC对样品进行细分，色谱条件为，色谱柱：Zorbax SB-C18 4.6*2505μm(AgilentUSA)，流动相：A液：乙腈/水：2:98(含三氟乙酸0.1％)；B液：乙腈/水：98:2(含三氟乙酸0.08％)；0–40％B大约3-6个柱体积，40–100％B 0.5-1个柱体积，100％B1-3个柱体积；流速：0.8ml/min；紫外检测器，检测波长为：280nm/258nm/214nm。；
    2)冻干的多肽样品重新溶解于Nano-RPLC Buffer A中，在线Nano-RPLC液相色谱在EksigentnanoLC-UltraTM 2D系统(AB SCIEX)进行，溶解后的样品以2μL/min的流速上样到C18预柱上(100μm×3cm，C18,3μm,
    Figure PCTCN2016112078-appb-100001
    )，保持流速冲洗脱盐10min。质谱采用TripleTOF5600系统(AB SCIEX)结合纳升喷雾III离子源(AB SCIEX,USA)，质谱采集到的原始wiff图谱文件，采用Protein Pilot Software v.4.5(AB SCIEX,USA)软件进行数据加工处理，得到蝎毒耐热合成肽见序列表SEQ ID No.1；
    (2)用固相化学合成方法制备蝎毒耐热合成肽；
    (3)高效液相色谱(HPLC)纯化在多肽合成过程中会产生一些与目标肽结构类似的杂肽，色谱条件如下：色谱柱：Inertsil ODS-SP 4.6mm*250mm，含0.1％三氟乙酸乙腈-含0.1％三氟乙酸水，紫外检测器，检测波长为214nm；
    (4)用质谱技术对合成多肽进行结构确认，检索分析，检出蝎毒耐热合成肽的氨基酸序列见序列表SEQ ID No.1。
  3. 权利要求1所述的蝎毒耐热合成肽在制备抗癫痫、阿尔茨海默病或帕金森病药物中的应用。
PCT/CN2016/112078 2016-08-08 2016-12-26 一种蝎毒耐热合成肽及其用途 WO2018028117A1 (zh)

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CN109232718B (zh) * 2018-11-09 2020-04-14 泰安市启航生物科技有限公司 一种合成肽sp2及其应用
CN111647051B (zh) * 2019-03-04 2022-02-01 中国医科大学 一种基于钠通道结构的短肽及其应用
CN111896651B (zh) * 2020-07-30 2021-04-23 山东大学 一种白眉蝮蛇蛇毒类凝血酶特征多肽及其应用
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