CN101139592A - Sequence and preparation method for novel signal conus O-superfamily toxin and uses thereof - Google Patents
Sequence and preparation method for novel signal conus O-superfamily toxin and uses thereof Download PDFInfo
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- CN101139592A CN101139592A CNA2007100288790A CN200710028879A CN101139592A CN 101139592 A CN101139592 A CN 101139592A CN A2007100288790 A CNA2007100288790 A CN A2007100288790A CN 200710028879 A CN200710028879 A CN 200710028879A CN 101139592 A CN101139592 A CN 101139592A
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- conotoxin
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- 238000000825 ultraviolet detection Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
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Abstract
The invention relates to a China South-sea Signal Conus O-super family toxin sequence, a preparing method and application of the sequence. A gene is got by separating from China South-sea Signal Conus toxin tube, and the nucleotide sequence is as that shown in the sequence table.
Description
Technical field
The present invention relates to the technology of preparing of a kind of South China Sea signal conus O-superfamily toxin gene Lt6.3 and encoded polypeptides sequence lt6c thereof and this peptide species, and this kind toxin is studied the application in ionic channel medicine and the analgesic exploitation in neurobiology.
Background technology
Cone shell belongs to Mollusca Gastropoda Conidae (Conidae), and majority is perched the shallow water along the coast in tropical ocean, and minority is in the ballow of several meters of depth of waters rice surplus 200, because of profile is taper shape or the taro shape is gained the name.Cone shell is the biology of comparison youth, fossil record proof Conus comes across the Eocene era (Eocene) the earliest, and the disappearance of the ocean predatism mollusk ammonite that (mesozoic) period in secondary era and land dinosaur become extinct has simultaneously promoted that objectively the first time of cone shell, large-scale species formed.The large-scale radiation second time of cone shell starts from Miocene Period (Miocene), lasts till basically now.Brand-new venom composition and the effectively evolution of venom e Foerderanlage are all followed in the formation of each cone shell species.
Cone shell has powerful natural evolution ability, and the whole world 500 kinds of cone shells of having an appointment just comprise hundreds of species in the single kind, and this makes cone shell become the most successful biology of evolution in the marine invertebrate.Cone shell is a zoophagous animal, preys on by venom, can be divided into ichthyophagy cone shell (piscivorous), food spiral shell cone shell (molluscivorous), carnivorism cone shell (vermivorous) according to its predation.Entomophagy cone shell most species accounts for about 70% of whole cone shell kinds.It is minimum that the ichthyophagy cone shell accounts for the cone shell sum, but toxicity is the strongest, and the cone shell of the report incident that causes death is that such cone shell is caused basically, as picture-weaving in silk cone shell (C.textile), ground-tint cone shell (C.geographus) etc.
The cone shell venom is predation and phylactic primary armament, and the mixing toxin of the cocktail sample that it is made up of many single phallotoxins is called conotoxin (Conotoxin).Conotoxin can act on specifically neural valtage-gated and ligand-gated ion channel without hypotype and mediator acceptor, therefore be widely used in neurobiology research.Conotoxin is generally the micromolecule polypeptide of being made up of 7～41 amino-acid residues.Be rich in halfcystine mostly, have the disulfide linkage skeleton of high conservative.Compare (about 40～80 amino acid) with the toxin of many animals such as spider, scorpion, snake, sea anemone, the peptide chain much shorter of conotoxin is rich in disulfide linkage, and molecular structure is more tight, and biological activity is higher.Most of conotoxins are encoded by single mRNA, and the primary translation product is a kind of specific polypeptide precursor, are about 70-120 amino-acid residue, obtain mature peptide behind protease hydrolysis.
The early 1980s, the U.S. still its university Olivera BM scholar laboratory has carried out the comprehensive systematic study work of conotoxin the earliest.Conotoxin is little with its molecule, Stability Analysis of Structures, and wide and active strong characteristics have leapt in the first place of animal nerve toxin study to the receptor acting scope.Because their action target spots are extensive, can high special ground in conjunction with the acceptor and the various ionic channel of cytolemma epineural mediator and kassinin kinin, on the one hand, can directly be developed to medicine or be applied to clinical as the lead compound of new drug; On the other hand, can become and find to identify novel receptor in the neurobiology, study the important probe of acceptor structure activity relationship and regulating cell molecule mechanism thereof, and promoted the Study on Evolution of ionic channel.
Conservative estimation has conotoxins different more than 50000 kinds to exist, and having obtained isolating conotoxin so far has nearly thousand kinds, and tens of kinds of conotoxins have been applied for United States Patent (USP).They are with a wide range of applications in analgesia, ischemic protection, epilepsy therapy, some medical diagnosis on disease and acceptor research, have enter clinical study or by the FDA official approval for the treatment new drug, as specific diagnostic reagent and anodyne.At present, ω-CTX M VIIA (the SNXIII that develops by Elan company, trade(brand)name: Ziconotide), because of it directly acts on the N-type calcium channel that is distributed in nervous tissue, need not second messenger or albumen, habituation has not become the medicine of new generation for the treatment of intractable neuralgia, passed through the III clinical trial phase, formally by FDA approval listing.And another compd A M336 effect that is derived from ω-CTX C VID is similar to Ziconotide, because of its selectivity to the N-calcium channel stronger, side effect is lower, has ratified to enter clinical experimental stage as the medicine of the serious anti-morphineation chronic pain of antagonism.In addition, Conantokin-G is as the antagonist of nmda receptor high selectivity, and is effective to the epilepsy that is difficult to treat, and also finished the I clinical trial phase.Other directions of the medicinal research of conotoxin also have: have the inhibiting T-superfamily conotoxin of norepinephrine transporter, can be used for treating dysthymia disorders.And some conotoxins that suppress α 1-adrenoceptor, can be used for treating the urinary incontinence that the benign prostate hyperplasia causes.Meanwhile, round the stability that strengthens drug molecule, reduction anaphylaxis and increase solvability are beneficial to the molecular modification research work of oral purpose and also carry out.
On the other hand, conotoxin is as the fabulous probe or the instrument of research ionic channel and membrane receptor, become the tool master of valtage-gated type calcium channel (VSCCs) and N type acetylcholine receptor passages such as (nAChRs), acceptor evaluation and diagnosis, obtained in the neuropharmacology field using widely.
Cone shell has extensive distribution in marine site, China South Sea, and kind surplus identified cone shell has 80 approximately mainly is distributed in Taiwan, Guangdong, Guangxi, Hainan Zhu Sheng and the Xisha and the Nansha Islands etc.The research work of aspects such as domestic in recent years biological chemistry of also carrying out conotoxin, molecular biology and pharmacological action.The people such as Chen Jisheng researcher of Beijing pharmaceutical chemistry institute of the Chinese Academy of Sciences in class cone shells such as barrel-shaped cone shell (C.betulinus), unique cone shell (C.caracteristileus), calamus cone shell (C.vexillum), ground-tint cone shell isolation identification the conotoxin of the new sequence of kind surplus ten.The Lu Baisong of BIO ENGINEERING INST MILITARY, Huang Peitang and Dai Qiuyun etc. have also found toxin composition and the gene order that some are new from the strain line cone shell of China, picture-weaving in silk cone shell.Peng of tropical agriculture university generation is clear, Russell orchid etc. from calamus cone shell genome, be cloned into two new conotoxin genes (GenBank registration number AY316159, AY316160); The marble cone shell (C.marmoreus) that also produces from Hainan, unreal cone shell (C.magus), conus (C.littertus), warrier cone shell (C.miles), unique cone shell, picture-weaving in silk cone shell, barrel-shaped cone shell, defect punt-pole cone shell (C.lividus) etc. have found to have the 35 kinds of O-superfamily conotoxins and the gene thereof of medicinal function totally 12 kinds respectively simultaneously.
Though there is abundant cone shell resource in China, owing to start late, the research for conotoxin at present still is in initial stage, and wide research space and DEVELOPMENT PROSPECT are arranged.
Because conotoxin differs greatly at its gene order and protein structure and function aspects, have of a great variety, complex structure and height genetic diversity feature.Make up conus poison pipe cDNA library and can systematically study toxin composition, the express spectra of carnivorism cone shell, find the new distinctive toxin sequence of carnivorism cone shell.Up to now, made up the cDNA library of multiple cone shell abroad, the South China Sea conus is a kind of entomophagy cone shell, does not still have the report about its toxin study in the world.
In addition, the biochemical structure of at present relevant conotoxin and the molecular structure that the active research of neuropharmacology all is based on the natural extract toxin thereof are studied with artificial synthetic toxin.Though be cloned into a lot of conotoxin genes, rarely had the report of conotoxin gene vivoexpression at present.The reorganization conotoxin will replace the synthetic toxin that productive rate is low, cost is expensive with its of fine quality, inexpensive advantage.Carry out conotoxin vivoexpression Study on Technology, will have very important theoretical investigation value and using value.
Therefore, the biological chemistry, pharmacology of the carrying out China's South Sea signal conotoxin especially exploratory development of its molecular biology and gene engineering technology field will help further investigation to corresponding acceptor of new lps molecule and mechanism of action thereof, and the medicinal development and use that can be China's cone shell oceanic resources provide the most important theories foundation.
Summary of the invention
The object of the present invention is to provide a kind of new South China Sea signal conus O-superfamily toxin gene Lt6.3, its encoded polypeptides sequence lt6c and analogue and derivative.
Another object of the present invention is to provide the production method of this peptide species.
The 3rd purpose of the present invention is to provide with full cell patch tongs technology has investigated the restraining effect of this peptide species to rat DRG cellular sodium electric current, thereby for the instrument medicine of preparation neurobiology research with treat disease medicaments such as irregular pulse, Persistent Pain, epilepsy and apoplexy data are provided.
The 4th purpose of the present invention is to provide to test with the mouse hot plate and investigates this peptide species and whether have the central analgesia effect, thereby provides data for the preparation analgesic.
By the structure in cDNA library and the method for order-checking, from South China Sea conus poison pipe, separate obtaining O-superfamily conotoxin Lt6.3 gene, its cDNA sequence as in the sequence table＜400〉1 sequences shown in.
The polypeptide of the invention described above coded by said gene, its precursor peptide aminoacid sequence as in the sequence table＜400〉2 sequences shown in; The aminoacid sequence of its mature peptide as in the sequence table＜400〉3 sequences shown in; The molecular weight of its mature peptide is 2949.46 dalton.
This albumen by prokaryotic cell prokaryocyte fusion expression vector pTRX in intestinal bacteria with soluble formal representation in the born of the same parents.
(my laboratory makes up and applies for a patent described expression vector pTRX voluntarily, patent name is: a kind of efficient prokaryotic expression carrier, number of patent application: ZL 00124832.4) being is to merge the companion body with the Trx, the efficient expression vector of the affinity labelling site of middle insertion His and the recognition site of Protease F actorXa.
This recombination signal conotoxin lt6c energy blocking part sodium channel reduces the peak point current of sodium channel, but does not influence the gate characteristic of passage.
The selected conus of the present invention picks up from Sanya, Hainan Province.
The structure in conus poison pipe cDNA library: get breechblock alive, use the broken spiral shell shell of larynx lock, expose spiral shell meat, careful on ice and sharp separation poison tubing extracts total RNA.Reverse transcription is a synthetic double chain cDNA behind the chain cDNA, and double-stranded cDNA is connected back Transformed E .coli with the pMD18T carrier, and preserves each recombinant clone.
The present invention has therefrom obtained the clone of 1 new coding conotoxin by to the extensive sequencing of library recombinant clone, called after Lt6.3 (its dna sequence dna as in the sequence table＜400〉1 sequences shown in).New 72 amino-acid residues of genes encoding, 28 amino-acid residues of mature peptide coding, mature peptide segment molecule amount is 2949.46 dalton.
The present invention goes up (this experiment structure) with the mature peptide gene clone of the new toxin lt6c of coding to prokaryotic fusion expression vector pTRX by having designed a pair of primer, construction expression plasmid and with its transformed into escherichia coli BL21 (DE3).This expression vector (pTRX-lt6.3) is promotor with T7, TRX is that molecule merges the companion body, helps recombinant protein correctly folding, with soluble formal representation, the C end of TRX has gentle sequence and 6 * His structure, is convenient to utilize immobilization metal part affinity chromatography to carry out purifying.Designed upstream primer contains Protease F actorXa site, is beneficial to the monomeric acquisition of foreign protein.By to incubation time, induction time, the groping and optimize of conditions such as temperature, TRX-lt6.3 Expression of Fusion Protein amount is higher, and the overwhelming majority is in solvable state.
The electricity Physiological Experiment is the result confirm, recombination signal conotoxin lt6c i.e. obvious blocking part sodium-ion channel under the 0.1 μ M concentration, reduce the peak point current of sodium channel, can be used as the research and development that probe is used for the classification evaluation of ionic channel type and hypotype or is used for the instrument medicine, be used to prepare the instrument medicine of neurobiology research and the medicine of diseases such as treatment irregular pulse, Persistent Pain, epilepsy and apoplexy.
Mouse hot-plate test result confirms that the middle and high dosage group of recombination signal conotoxin lt6c is compared with the blank group, has significant difference (P＜0.05, P＜0.001); Can think tentatively that thus the middle and high dosage group of lt6c has certain analgesic activity, thereby provide data for preparing analgesic.
Expression plasmid clone method of the present invention:, use CaCl with reference to Sambrook (Sambrook, et al.1989, Molecularcloing.Cold Spring Harbor Labroratory Press.USA) method
2Method with plasmid Transformed E .coli.DH5
Or BL21 (DE3) bacterial strain, with containing penbritin (10
G/mL) LB culture medium culturing transforms bacterial strain, and alkaline process extracts plasmid.
Description of drawings
Fig. 1 is the SDS-PAGE electrophorogram behind the fusion rotein TRX-lt6c abduction delivering
1: protein molecular weight standard; 2: the total protein of intestinal bacteria abduction delivering; 3: the total protein that changes 24 ℃ of abduction deliverings of intestinal bacteria of plasmid pTRX-Lt6.3 over to; 4: the supernatant that changes 24 ℃ of abduction deliverings of intestinal bacteria of plasmid pTRX-Lt6.3 over to; 5 change the precipitation of 24 ℃ of abduction deliverings of intestinal bacteria of plasmid pTRX-Lt6.3 over to
Fig. 2 is fusion rotein TRX-lt6c Ni
2+The protein electrophoresis figure of affinity chromatography wash-out
1:Ni
2+Affinity chromatography penetrates the peak; 2:50 ~ 75mM imidazoles elution peak; 3:75 ~ 100mM imidazoles elution peak; 4:150 ~ 175mM imidazoles elution peak; 5:175 ~ 200mM imidazoles elution peak; 6:＞200mM imidazoles elution peak; 7: the level pad elution peak; 8:500mM imidazoles elution peak; 9: protein molecular weight standard
Fig. 3 is the fusion rotein TRX-lt6c Ni after optimizing
2+The SDS-PAGE electrophorogram of affinity chromatography elution peak
1: protein molecular weight standard; 2:TRX-lt16c Ni
2+Affinity chromatography penetrates the peak; 3:TRX-lt16c75mM imidazoles elution peak; 4:TRX-lt16c 180mM imidazoles elution peak
Fig. 4 is the chromatographic peak figure of fusion rotein TRX-lt6c through the G25 gel-filtration
The design sketch that Fig. 5 cuts through factor Xa enzyme for fusion rotein TRX-lt6c
1: protein molecular weight standard; 2: fusion rotein TRX-lt6c; 3:20 ℃ of enzyme cut fusion rotein TRX-lt6c 16 hours; 4: albumen lt6c
Fig. 6 is a recombinant protein lt6c Sephadex G50 sieve chromatography collection of illustrative plates
1: peak 1 factor Xa enzyme and foreign protein; 2: peak 2TRX; 3: peak 3 reorganization conotoxin lt6c; 4: compositions such as peak 4DTT and enzyme cutting buffering liquid
Fig. 7 is the efficient liquid phase chromatographic analysis figure of recombinant protein lt6c
Fig. 8 is the electrospray ionization mass spectrum analysis chart at recombinant protein lt6c RPLC peak
Fig. 9 is the flying time mass spectrum analysis figure of recombinant protein lt6c
Figure 10 is the influence of recombinant protein lt6c to rat dorsal root ganglion cell Na channel current
Wherein, Figure 10 A is-80mV for clamping down on voltage, and stimulation voltage is from-80mV to+30mV, and the recurrent interval is 10mV, and the pulse width of stimulation voltage is under the condition of 15ms, contrasts and add the variation of 800nM lt6c after-current peak shape
Figure 10 B is the I-V curve, the variation of peak current after having shown contrast and having added 800nM lt6c 20min
Figure 11 is the analgesia drug effect NS on the recombinant protein lt6c physiological models: the blank group; H:lt16c sample high dose group; Dosage group in the M:lt16c sample; L:lt6c sample low dose group
Embodiment
Following examples will help those of ordinary skill in the art further to understand the present invention, but not limit the present invention in any form.
Embodiment 1: the structure and the evaluation in conus poison pipe cDNA library
The extraction of total RNA is synthetic with cDNA: separates South China Sea conus poison pipe, carry out poison with reference to the TRIZOL LS reagent specification sheets of Gibco BRL company and manage total RNA extraction.Get the total RNA of 1 μ g conus poison pipe with SMART III olignuclotide (5 '-AAGCAGTGGTATCAACGCAGAGTGGCCATTATGGCCGGG-3 ') and CDSIII/3 ' PCR primer (5 '-ATTCTAGAGGCCGAGGCGGCCGACATG-d (T)
30N
-1N-3 ') carries out synthetic first chain of reverse transcription, obtain 10 μ lcDNA, the first chain product.
The structure and the evaluation in conus poison pipe cDNA library: get cDNA
Be used for ligation, transform the back coated plate.From flat board the picking mono-clonal protect kind and at random the mono-clonal of picking certain number check order and bioinformatic analysis.
Embodiment 2: the sequential analysis of conotoxin gene Lt6.3
The plasmid of South Sea signal conus neurotoxin gene Lt6.3 and sequence are from the est sequence of being cloned in the South China Sea conus poison pipe cDNA library.72 amino-acid residues of precursor peptide coding of South Sea signal conus neurotoxin gene Lt6.3,28 amino-acid residues of mature peptide coding.
Utilize BLAST N and BLAST X (http://www.ncbi.nlm.nih.gov/BLAST) algorithm in public databases such as existing GenBank, survey Nucleotide and derived protein sequence to be carried out the homology retrieval.Potential reading frame (ORFs) utilizes SEQTOOLS8.0 to locate.The homology of sequence relatively adopts CLUSTAL X software to carry out.The protein signal peptide sequence prediction is undertaken by website http://www.cbs.dtu.dk/services/SignalP.Proteinic molecular weight, theoretical iso-electric point, charge distribution and stability parameter etc. are finished by ProtParam tool (http://www.expasy.org/tools/protparam.html) software.The prediction of tertiary structure be by SWISS-MODEL (
Http:// swissmodel.expasy.org//SWISS-MODEL.html) the homology mould program of building finishes, the PDB file of generation is analyzed by Swiss-PdbViewer software.
Embodiment 3: the structure of recombination signal conus O-superfamily toxin gene Lt6.3 expression plasmid
Nucleotide sequence (attgagggcgctggccgtgcaaggttgccggtagtccttgtggtctggttagtgaa tgctgcggaacttgcaatgttttacgcaatcgttgtgtg) according to the Lt6.3 gene encoding mature peptide, (my laboratory makes up voluntarily and applies for a patent in conjunction with the pTRX carrier, patent name is: a kind of efficient prokaryotic expression carrier, number of patent application: ZL 00124832.4) multiple clone site and restricted type endonuclease digestion site analytical results design four sections Oligo (table 1) of two genes.Added the recognition site of the disconnected and Protease F actorXa of the right side flap enzyme section of restriction enzyme Kpn I at 5 ' end of goal gene, and that 3 ' end has added the left side flap enzyme section of terminator codon and Restriction Enzyme NotI is disconnected.
According to above-mentioned requirements design nucleotide sequence, and synthetic by Shanghai Bo Ya company, and dry powder is standby-20 ℃ of preservations, faces with before being diluted to 20 μ mol/L.
Table 1 is used to encode and waits to express four sections nucleotide sequences of conotoxin gene Lt6.3
The fragment of using the KpnI/NotI restriction enzyme to cut marks with underscore, and Xa factor protease digestion place marks with shade, and " * " represents terminator.
The phosphorylation of nucleic acid fragment, annealing and be connected.Synthetic four segment DNA segments are become with the atom level water dissolution
Solution.Four oligo segments of synthetic are divided into two groups of complementary pairing and anneal respectively and phosphorylation.Get complementary pairing nucleic acid each
In 94 ℃ of water-baths, be incubated 2min, in water-bath, naturally cool to 45 ℃ then; Set
The phosphorylation reaction system adds deionized water successively in the good solution of above-mentioned annealing
The Starch phosphorylase damping fluid
ATP
, the T4 polynucleotide kinase
37 ℃ of temperature were bathed 2 hours behind the mixing.
With the T4DNA ligase enzyme two groups of oligo segments are connected with the pTRX carrier.Make up fusion expression plasmid pTRX-Lt6.3.Recombinant plasmid is carried out two-way order-checking, and its sequence and synthetic template sequence result are in full accord, illustrate that the reading frame of the Lt6.3 gene that inserts expression vector is correct.
Embodiment 4: the expression of recombination signal conotoxin gene Lt6.3
Recombinant expression plasmid pTRX-Lt6.3 is transformed expressive host bacterium BL21 (DE3) be built into engineering strain.Picking engineering strain list colony inoculation is in Amp
+LB liquid enriched medium in, 37 ℃ of thermal agitation overnight incubation are as kind of a daughter bacteria.Get kind of daughter bacteria and be inoculated in fresh Amp by 1: 100 volume ratio
+In the LB rich medium, 37 ℃ of thermal agitation amplification culture are to OD
600Be about at 0.8 o'clock, add IPTG to final concentration be 0.1mmol/L, add 20% glucose simultaneously to final concentration 0.2%, in 20 ℃ of abduction delivering 10hr.4 ℃, the centrifugal 10min of 5000rpm receive bacterium, and reservation 1ml bacterium liquid is used for the SDS-PAGE analysis.Behind TE damping fluid (pH8.0) the washing thalline with precooling, use the 50mmol/L Tris-HCl (pH8.0) of precooling again, the resuspended thalline of 500mmol/LNaCl solution.Adopt the power of JY92-II type ultrasonic cell disruptor (Ningbo Xin Zhike device Research Institute) with 600W, ultrasonic 4 circulation (ultrasonic 4s under the ice bath, 4s intermittently, repeating 90 times is a circulation) broken bacterial cell, get the expression that cleer and peaceful precipitation is carried out SDS-PAGE analyzing and testing recombinant protein respectively behind 4 ℃, the centrifugal 30min of 12000rpm.(see figure 1)
Embodiment 5: the purifying of recombination signal conotoxin gene Lt6.3 expressing protein
With the nickel affinity chromatography post of handling well, with ultrasonic damping fluid (50mM Tris, 500mM NaCl, pH8.0) 5 times of column volumes of balance.Express 4 ℃ of centrifugal receipts bacterium of intestinal bacteria of pTRX-Lt6.3, weigh, add the resuspended thalline of the ultrasonic damping fluid of 10ml in the wet bacterium of every 1g, carrying out ultrasonic bacteria breaking, centrifugal ultransonic sample is got supernatant liquor and is carried out Ni
2+-Chelating Sepharose Fast Flow affinity chromatography.Detect with 215nm and two ultraviolet detection wavelength of 280nm.After sample is eluted to baseline with level pad, collects and pass protein peak, the elutriant with different imidazole concentrations carries out wash-out successively, collects elution peak, carries out Tricine SDS-PAGE and analyzes (see figure 2).Fusion rotein can not eluted under the 100mM imidazole concentration, and imidazole concentration almost can elute whole target proteins when the 200mM left and right sides.Therefore, in our purge process after amplification, adopt the method for two step wash-outs to obtain purer recombination fusion protein.(see figure 3)
Embodiment 6: recombination signal conotoxin lt6c enzyme is cut the back purifying
Pass through Ni
2+The fusion rotein of the resulting purifying of Chelating Sepharose Fast Flow affinity chromatography is crossed Sephadex G-25 gel column and is changed (see figure 4) behind the enzyme cutting buffering liquid, carries out proteolytic enzyme factor Xa enzyme enzyme and cuts.TRX-lt6c is at 21 ℃ of cutting 16hr, and target protein can cut fully.(see figure 5)
Adopted Sepharose G50 molecular sieve gel chromatography to separate, and detected the ultraviolet absorption value of elutriant at 280nm and 215nm place wavelength.Through Sepharose G50 gel permeation chromatography, we detect four ultraviolet absorption peaks, judge according to experience, and be the FactorXa enzyme successively according to these four peaks of priority of appearance time, TRX, conotoxin lt6c and enzyme are cut the micromolecular compound in the system.(see figure 6)
Because the conotoxin concentration of getting off from molecular sieve is very low, thereby in the experiment the 3rd elution peak carried out necessary concentrating through the 1kDa ultra-filtration membrane, directly carry out the separation that is further purified of RPLC then.The high performance liquid chromatography chromatography has adopted water and acetonitrile moving phase system, and has added 0.1%TFA as the hydrophobicity of modification property ion pair material with the increase sample in system.Experimental result shows, along with increasing progressively of acetonitrile concentration in the moving phase, by detecting the ultraviolet absorption value at 215nm place, can obtain some little assorted peaks of a tangible elution peak and front and back.(see figure 7)
Embodiment 7: the target protein peak after the molecular weight that electron spray(ES) and flight time mass spectrum are identified reorganization conotoxin Lt6c and purity are got high performance liquid chromatography and separated carries out electron spray(ES) and flight time mass spectrum is identified its molecular weight.The result shows, retention time is that (seeing Fig. 8,9) matches for the molecular weight (predicting MW=2949.46) of molecular weight and the little peptide of purpose of component (lt6c) in the elution peak of 21.2min.Therefore, by being further purified of RPLC, little peptide and fusion partner have obtained separating preferably successively by the wash-out that dissociates.
Embodiment 8: recombination signal conotoxin lt6c is to the effect of sodium-ion channel
With SD rat dorsal root ganglion cell is experimental specimen, uses the record that full cell patch pincers pattern is carried out full cell currents.Control group and experimental group write down respectively is to use the full cellular sodium electric current under gradient depolarize condition before and after the different concns toxin.Clamp down on voltage and be-80mV, stimulation voltage is from-80mV to+30mV, and the recurrent interval is 10mV, and the pulse width of stimulation voltage is 15ms.Experiment all repeats more than 3 times.Figure 10 is indicated in and adds the DRG full cellular sodium electric current changing conditions of 0.8 μ M toxin (lt6c) front and back under gradient depolarize condition.
From experimental result picture as can be seen, the peak point current behind the adding toxin all has tangible reduction, but the peak shape of sodium current does not significantly change.The recombinant expressed conotoxin of this explanation can the blocking part sodium channel, and does not influence the gate characteristic of passage.
According to the source recording figure of full cell currents, with stimulation voltage sodium current peak value under this voltage is mapped, draw current-voltage curve, shown in Figure 10 B.The result shows, under the effect of respective concentration toxin, the dependence of rat dorsal root ganglion cellular sodium electric current and voltage and the activation potential of sodium channel do not change, the current potential that peak point current produces (20 or-30mV) and upset current potential (20 or 30mV) all not have to take place obviously change.Meanwhile, obviously the blocking part cell is interior to sodium current (n=3) for toxin.From above analysis as can be seen, the recombinant toxin molecule does not change the activation of passage and the kinetic curve of inactivation in the blocking-up sodium channel.Further experiment shows that also this blocking effect has concentration dependent.
Embodiment 9: the central analgesia effect experiment method on the physiological models
General planning: mouse hot plate method
Choosing NIH is 100 of mouse, body weight
Complete is female.Earlier mouse is screened before the experiment, mouse is put on the hot plate pain threshold detector that is heated to 55 ℃ in advance,, measure 2 times from dropping into hot plate to occurring licking the threshold of pain of the time of metapedes for this mouse with stopwatch record mouse, picking out the mouse that threshold of pain is no more than 20s is that eligible is formally tested.Mouse after the screening is divided into 4 groups at random, i.e. blank group, the basic, normal, high dosage group of lt6c sample, 10 every group.Carry out intraperitoneal injection during the experiment beginning, wherein the basic, normal, high dosage group of lt6c sample gives dosage and is respectively 0.07mg/kg, 0.14mg/kg, and 0.28mg/kg, the blank group gives isopyknic physiological saline.0.5h, 1h, 1.5h, 2h, 3h measure once behind drug administration by injection, surpass 60s as threshold of pain and then press 60s calculating (the attention time should not be too of a specified duration in order to avoid scald the mouse foot).After experiment finishes, carry out statistical procedures and calculate threshold of pain raising percentage.Percentage=(the preceding average threshold of pain of average threshold of pain-medication after the medication) the preceding average threshold of pain of ÷ medication * 100% is improved in the threshold of pain.
Can be got by experimental result, the threshold of pain before and after the administration of blank group mouse is basicly stable, and prompting is better to the tolerance of thermal stimulus reaction through the mouse after screening.All in various degree the raisings of threshold of pain after the basic, normal, high dosage group mouse administration of given the test agent lt6c, wherein during administration 0.5h, the threshold of pain of three dosage group mouse of given the test agent lt6c with the same period blank group compare, have significant difference (P＜0.01, P＜0.001); When administration 1h and 2h, the threshold of pain of the middle and high dosage group mouse of given the test agent lt6c with the same period blank group compare, have significant difference (P＜0.01, P＜0.001); During administration 3h, the threshold of pain of given the test agent is reduced to minimum, and wherein the middle and high dosage group of lt6c is compared with the blank group, has significant difference (P＜0.05, P＜0.001); Can think tentatively that thus the middle and high dosage group of lt6c has certain analgesic activity.(seeing Figure 11)
Sequence table
＜110〉Zhongshan University
＜120〉a kind of new signal conus O-superfamily toxin sequence, preparation method and application thereof
<160>3
<210>1
<211>462
<212>DNA
＜213〉South China Sea signal line cone shell (Conus litterarus)
<400>1
gacctcgtct?gcctgatcct?tacacggcga?gcctgacttc?acctttcttc?gctgcctcct?60
ttggcatcac?ccagaccatc?atcagaatga?aactgacgag?tgtggtgatc?gtcgctgtgt?120
tgttcctggc?ggcctgtcaa?ctcactacat?ctgatggctc?cagaggtacg?tggaaggatc?180
gtgctgtgag?gtcgatcacc?aaagtctcca?tgttgcgatg?gccctgcaag?gttgccggta?240
gtccttgtgg?tcttgttagt?gaatgctgcg?gaacttgcaa?tgttttacgc?aatagatgtg?300
tgtgagtggc?tgatccggcg?tctggtcttt?ccgccttctg?tgctctatcc?ttgttcgcct?360
gcgtcctcca?tagctgtgag?tggtcatggg?ccactcaaca?cctactcctc?tggaggcttc?420
agaggaacta?cattcaaata?aagccgcatt?gcaatgaaaa?aa 462
<210>2
<211>72
<212>PRT
＜213〉South China Sea signal line cone shell (Conus litterarus)
<400>2
Met?Lys?Leu?Thr?Ser?Val?Val?Ile?Val?Ala?Val?Leu?Phe?Leu?Ala
1 5 10 15
Ala?Cys?Gln?Leu?Thr?Thr?Ser?Asp?Gly?Ser?Arg?Gly?Thr?Trp?Lys
20 25 30
Asp?Arg?Ala?Val?Arg?Ser?Ile?Thr?Lys?Val?Ser?Met?Leu?Arg?Trp
35 40 45
Pro?Cys?Lys?Val?Ala?Gly?Ser?Pro?Cys?Gly?Leu?Val?Ser?Glu?Cys
50 55 60
Cys?Gly?Thr?Cys?Asn?Val?Leu?Arg?Asn?Arg?Cys?Val
65 70
<210>3
<211>28
<212>PRT
＜213〉South China Sea signal line cone shell (Conus litterarus)
<400>3
Trp?Pro?Cys?Lys?Val?Ala?Gly?Ser?Pro?Cys?Gly?Leu?Val?Ser?Glu
1 5 10 15
Cys?Cys?Gly?Thr?Cys?Asn?Val?Leu?Arg?Asn?Arg?Cys?Val
20 25
Claims (6)
1. from South China Sea conus poison pipe, separate obtaining a kind of gene, its nucleotide sequence as in the sequence table＜400〉1 sequences shown in.
2. a peptide species, it contains aminoacid sequence or its conservative property variation peptide sequence or its reactive derivative of the described genes encoding of claim 1.
3. by the described polypeptide of claim 1, it is characterized in that: this amino acid sequence of polypeptide is as sequence table＜400〉shown in 2.
4. the mature peptide fragment of the described polypeptide of claim 3, its aminoacid sequence is as sequence table＜400〉shown in 3.
5. the production method of the described polypeptide of claim 4, carry out according to following steps:
(1) will encode the described mature peptide gene clone of claim 4 to prokaryotic fusion expression vector pTRX, construction expression plasmid and with its transformed into escherichia coli BL21 (DE3);
(2) e. coli bl21 (DE3) after cultivation transforms;
(3) e. coli bl21 (DE3) is carried out ultrasonic degradation, with the lysate of expression product through Ni
2+Chelating Sepharose affinity chromatography obtains fusion rotein;
(4) fusion rotein is through factor Xa proteolytic enzyme cutting, Sepharose G50 molecular sieve gel chromatography and RPHPLC (reversed-phase high-performance liquid chromatography), the mature peptide of the lt6c polypeptide that obtains recombinating.
6. claim 2,3,4 described polypeptide can be used for the detection of ionic channel type, the application of neurobiology tool drug and analgesic.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101979572A (en) * | 2010-11-08 | 2011-02-23 | 中山大学 | Preparation for South China Sea conus striatus toxin S4.3 and application |
| CN102604960A (en) * | 2012-03-23 | 2012-07-25 | 中山大学 | Neurotoxin S10a of South China Sea Conus striatus, coding sequence and application of the neurotoxin |
| CN105907763A (en) * | 2016-04-21 | 2016-08-31 | 中山大学深圳研究院 | South China Sea conotoxin encoding sequence and application thereof |
| CN113243338A (en) * | 2021-05-14 | 2021-08-13 | 福州大学 | Construction and evaluation method of mouse ischemic stroke model |
-
2007
- 2007-06-28 CN CNA2007100288790A patent/CN101139592A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101979572A (en) * | 2010-11-08 | 2011-02-23 | 中山大学 | Preparation for South China Sea conus striatus toxin S4.3 and application |
| CN102604960A (en) * | 2012-03-23 | 2012-07-25 | 中山大学 | Neurotoxin S10a of South China Sea Conus striatus, coding sequence and application of the neurotoxin |
| CN102604960B (en) * | 2012-03-23 | 2013-07-10 | 中山大学 | Neurotoxin S10a of South China Sea Conus striatus, coding sequence and application of the neurotoxin |
| CN105907763A (en) * | 2016-04-21 | 2016-08-31 | 中山大学深圳研究院 | South China Sea conotoxin encoding sequence and application thereof |
| CN113243338A (en) * | 2021-05-14 | 2021-08-13 | 福州大学 | Construction and evaluation method of mouse ischemic stroke model |
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