CN102190708B - Barrel-shaped alpha conantokin Bt1.3 and application thereof - Google Patents

Abstract

The invention discloses barrel-shaped alpha conantokin Bt1.3 and application thereof. The amino acid sequence of the barrel-shaped alpha conantokin Bt1.3 is a sequence 2 in a sequence table; and the nucleotide sequence of a coding gene of the conatokin is a sequence 1 in the sequence table. The connection modes of a disulfide bond of the conantokin are Cys1-Cys3 and Cys2-Cys4; and Cys starting from the amino terminal of the conantokin is respectively marked as Cys1, Cys2, Cys3 and Cys4 in sequence. Experiments prove that: Bt1.3 linear peptide (RKCCSNPACNRYNPAIC) is synthesized by an Fmoc-solid-phase peptide synthesis method, and is folded by one step in buffer solution of NH4HCO3 to form the Bi1.3 conantokin; and analgesic experiments prove that the alpha conantokin has high analgesic activity in rat sciatic nerve hemisection models.

Description

A kind of barrel-shaped α conotoxin peptide Bt1.3 and application thereof

Technical field

The present invention relates to biological technical field, relate in particular to a kind of barrel-shaped α conotoxin peptide Bt1.3 and application thereof.

Background technology

Neuropathic pain is a kind of by central or peripheral nervous system primary pathology or dysfunction and the pain syndromes that causes.The classical symptom of neuropathic pain comprises paresthesia, hyperpathia and allodynia etc.Present many major diseases (as cancer, acquired immune deficiency syndrome (AIDS), traumatic nerve injury, leprosy, sclerosis, zoster) all can cause the damage of nervous function and cause neuropathic pain, medicine commonly used mainly contains the coupling of anticonvulsive drug (carrying western phthalein, oxcarbazepine as Lay), opiates medicine (as morphine etc.) and various thymoleptic clinically, but the problems such as the tolerance of bringing due to toxic side effect and long-term prescription, habituation, result for the treatment of is not good.Therefore, the analgesic of Development of New Generation high reactivity, low tolerance and non-habituation is very necessary.Current research shows: neuron pattern hypotype nAChRs α 9 α 10, α 7 and α 3 β 2 are relevant to analgesia, are that development is without the desirable target spot that causes the chronic analgesic of addiction.

(α-CTX) is a class polypeptide that is purified into from cone shell poison gland pipe the earliest to alpha-conotoxin, belong to the A superfamily, generally formed by 9-18 amino acid, contain 2 pairs of disulfide linkage, skeleton construction is CCXmCXnC (C represents halfcystine, and X represents non-halfcystine, and m and n represent the quantity of non-halfcystine), according to the difference of m and n, α-CTX is divided into the subfamilies such as α 3/5, α 4/3, α 4/6, α 4/7.Wherein α 3/5, and as α-MI, Main Function is in vertebrate muscularity acetylcholine receptor, and other hypotypes optionally act on the different subtype (as α 3 β 2, α 9 β 10 etc.) of vertebrate nervous system type acetylcholine receptor mostly.Aspect analgesic activities research, find that at present Rg1A, Vc1.1, three kinds of α-conotoxin peptides of MII have analgesic activities (Callaghan B et al.J Neurosci, 2008 preferably; 28:10943-10951; Young T et al.Brain Res.2008; 1229:118-124.), can alleviate the pain that causes due to wound, inflammation or nervosa damage.The analgesic activity target spot of α conotoxin peptide Rg1A, Vc1.1 may be acetylcholine receptor alpha 9 α 10 hypotypes (Vincler et al.PNAS.2006,103 (46), 17880-17884), and the action target spot of MII is acetylcholine receptor alpha 3 beta 2 subunit types (Young Y, et al.Brain Res., 2008,1229:118-124).Act on peripheral nervous system, can adopt that polypeptide is subcutaneous, the administering modes such as muscle or abdominal cavity, the convenient application.The current research of Vc1.1 target for pain management shows, Vc1.1 be the inhibition of the N-type calcium channel that mediates by GABAB bring into play analgesic activity (Klimis H et al.PAIN 2011,152:259-266), but also might be relevant to present undiscovered mechanism.

Summary of the invention

An object of the present invention is to provide a kind of barrel-shaped α conotoxin peptide Bt1.3.

Polypeptide provided by the invention, its aminoacid sequence are the sequence 2 in sequence table.

The encoding gene of described polypeptide is also the scope of protection of the invention.

The encoding gene of described polypeptide is the DNA molecular of following (1) or (2) or (3):

(1) DNA molecular shown in sequence 1 in sequence table;

(2) DNA molecular that the DNA sequence dna hybridization that limits with (1) under stringent condition and coding have the identical function polypeptide;

(3) DNA sequence dna that limits with (1) has 70% at least, have at least 75%, have at least 80%, have at least 85%, have at least 90%, have at least 95%, have at least 96%, have at least 97%, have at least 98% or have at least a DNA molecular that 99% homology and coding have the identical function polypeptide.

Above-mentioned stringent condition can be at 6 * SSC, and in the solution of 0.5%SDS, hybridization, then use 2 * SSC under 68 ℃, and 0.1%SDS and 1 * SSC, 0.1%SDS respectively wash film once.

The mode of connection of the disulfide linkage of described polypeptide is Cys1-Cys3 and Cys2-Cys4; The Cys that begins from the aminoterminal of described polypeptide is denoted as respectively Cys1, Cys2, Cys3, Cys4 in order.

The application of described polypeptide in preparing the analgesic product is also the scope of protection of the invention.

Described product is medicine.

Utilize conserved regions and 3 ' the RACE cloning process of α conotoxin signal peptide, the clone obtains new α 4/7 a type conotoxin peptide Bt1.3 gene (Fig. 1) from barrel-shaped cone shell (Conus betulinus) the poison gland pipe in Chinese Xisha Islands of South China Sea marine site, the NCBI sequence alignment the analysis showed that this conotoxin peptide has brand-new amino acid and forms, and has significant difference with the alpha-type conus polypeptide of present report on amino acid forms.

Utilize the Fmoc-solid phase method of peptide synthesis to synthesize Bt1.3 linear peptides (RKCCSNPACNRYNPAIC), then at NH ₄HCO ₃In damping fluid a step folding, product forms 2 peaks (I peak and II peak, Fig. 2 A).The disulfide linkage mode of connection of general natural α conotoxin is mainly " C1-C3, C2-C4 ", is ball-like structure, goes out the peak early in the reversed-phase HPLC analysis.Be further to confirm the disulfide linkage arrangement mode at the folding I of Bt1.3 peak, synthesized C1 and C3 position halfcystine with the linear peptides (RKC (Acm) CSNPAC (Acm) NRYNPAIC) of Side chain protective group Acm (ethanamide).The first step approximately forms the product (Fig. 2 B-b) that contains a pair of disulfide linkage (C2-C4) after 28h in the oxidation of the 0.1M of pH 7.7 Tris-HCl damping fluid, this product of purifying is also sloughed Acm and is obtained disulfide bond assignment and be the product III peak (Fig. 2 B-c) of (C1-C3, C2-C4) in iodine solution.Then the common sample introduction in two-step approach product III peak that a folding product I peak (Fig. 2 A) that obtains of step and disulfide linkage is known, result shows both retention time consistent (Fig. 2 B-d).The disulfide linkage arrangement mode at confirmation Bt1.3 folding I of one step peak is " C1-C3, C2-C4 ".Its specific charge of mass spectroscopy " M+1 " is 1907.87, and (theoretical value is 1907.81) conforms to calculated value.

Then, measure the analgesic activities of Bt1.3-I with rat sciatic nerve hemisection model (PSL), result shows that Bt1.3-I has very strong analgesic activities, the intramuscular injection very Bt1.3 of low dosage (0.3nmol/ only) can make Pain Regulation In The Rat improve approximately 26.7%, with suitable with the analgesic activities of dosage Vc1.1.In range of doses, the analgesic effect of this peptide presents dose-dependently (Fig. 3).

In addition, the action target spot of Bt1.3 is studied.With rat acetylcholine receptor alpha 3 β 2 and α 3 β 4 subtype expression on Xenopus Oocytes, measure Bt1.3-I to the restraining effect of hypotype vagusstoff excitation current with the two electrodes voltage clamp, result shows that Bt1.3 can act on α 3 beta 2 subunit types specifically, the Bt1.3-I of 1000nM can suppress neurons of rats α 3 beta 2 subunit type vagusstoff excitation currents approximately 65.3%, and to a little less than α 3 β 4 hypotype effects (Fig. 4).

The Bt1.3 polypeptide that the present invention of experiment showed, of the present invention synthesizes has very strong analgesic activities, this and its can act on specifically α 3 beta 2 subunit bases.

Description of drawings

Fig. 1 is the translation product of Bt1.3 precursor peptide cDNA sequence and prediction,

Underscore is depicted as signal peptide sequence, and dash area is the mature peptide sequence.

Fig. 2 is the detection figure of synthetic Bt1.3,

Fig. 2 A is that the folding product HPLC of Bt1.3 oxidation step analyzes; A) linear peptides; B) folding product I of step and an II; C) the product I after purifying.

Fig. 2 B is that folding product III of two step of Bt1.3 reaches and folding product I of a step sample introduction HPLC analysis altogether; A) linear peptides; B) the folding product of the first step; C) the folding product III of second step; D) biased sample of folding product I and III.

Fig. 3 is that after Bt 1.3-I administration, the 1.5h threshold of pain changes,

Histogram graph representation PNL rat muscle injecting normal saline, Vc1.1 and various dose Bt1.3, the average threshold of pain increase rate (%) after 1.5h.

Fig. 4 be Bt1.3 to the action effect of different nervous system type nAChR,

A)-c) be respectively 10nM, 50nM and 1000nM Bt1.3 to the inhibition of rat α 3 beta 2 subunit type ACh excitation currents.D) inhibition of 1000nM Bt1.3 to rat α 3 β 4 hypotype ACh excitation currents.

Embodiment

The experimental technique that uses in following embodiment is ordinary method if no special instructions.

In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.

Embodiment 1, α-conotoxin peptide Bt1.3's is synthetic

One, the gene clone of α-conotoxin peptide Bt1.3

1, cDNA's is synthetic

The barrel-shaped cone shell of live body (Conus betulinus) that gathers from Chinese Xisha Islands of South China Sea marine site is as sample material.Adopt the Trizol method to extract the total RNA of cone shell poison gland pipe.Use Dalian TaKaRa company 3 ' Full-RACE test kit (3 '-Full RACE Core Set Ver 2.0) to carry out the synthetic of cDNA, concrete grammar is as follows:

First add in proportion following component:

Mix, in 70 ℃ of sex change 10min, chilling 2min on ice.Then take this reaction solution as template, carry out reverse transcription by following reaction system and condition:

42 ℃ of reaction 1h, 70 ℃ of reaction 15min, reverse transcription product is in-20 ℃ of preservations.

2,3 ' of cDNA-RACE amplification

Signal peptide sequence design upstream outside primers F 1 and the inboard primers F 2 conservative according to conotoxin peptide A superfamily are carried out nest-type PRC with downstream outside primer R1 and the inboard primer R2 that test kit provides respectively.Each primer sequence is as follows:

F1：5’-ATG?GGC?ATG?CGG?ATG?ATG?TTC-3’

F2：5’-CTG?TTG?GTT?GTC?TTG?GCA?ACC?AC-3’

R1：5’-TAC?CGT?CGT?CGT?TCC?ACT?AGT?GAT?TT-3

R2：5’-CGCGGATCCTCCACTAGTGATTTCACTATAGG-3’

First round PCR, the cDNA that obtains in 1 is as template, and reaction system is as follows:

Reaction conditions is: 94 ℃ of denaturation 4min; 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 1min, totally 30 circulations; Extend 10min in 72 ℃ at last.

Second takes turns PCR, take first round PCR product as template, increases as the upstream and downstream primer with F2 and R2; Other components of reaction system, consumption and reaction conditions are with first round PCR.

3, PCR Product Identification and order-checking

After with 1% agarose gel electrophoresis, the PCR end product being carried out the initial gross separation evaluation, the purpose band is cut glue purification, (sky root biotech firm VT202-01), is transformed into bacillus coli DH 5 alpha then to be connected to the pGM-T carrier; Amicillin resistance screening transformant after bacterium liquid PCR identifies, is sent to order-checking with positive colony.

Result has the Nucleotide shown in sequence 1 in sequence table for this PCR end product, the aminoacid sequence of the polypeptide of this PCR product coding is as shown in the sequence 2 in sequence table, peptide sequence is to infer to come (as shown in Figure 1) according to precursor peptide and conotoxin characteristics, precursor peptide is to infer according to precursor-gene, in Fig. 1, underscore is depicted as signal peptide sequence, and dash area is the mature peptide sequence.This precursor peptide contains 62 amino acid, and wherein signal peptide sequence is comprised of 21 amino acid, and sequence signature is consistent with alpha-conotoxin, and the precursor peptide zone contains 21 amino acid, and mature peptide has the conservative halfcystine skeleton CCX of alpha-conotoxin ₄CX ₇C is therefore it is a kind of novel α 4/7 conotoxin.With the polypeptide called after Bt1.3 that clones.

Two, the synthetic and determination of disulfide bond of α conotoxin peptide Bt1.3

1, synthetic (" folding " is that finger-type becomes disulfide linkage, and the polypeptide after folding just has analgesic activities) of Bt1.3

(1) linear peptides is synthetic

With the synthetic Bt1.3 linear peptides (RKCCSNPACNRYNPAIC) of ABI 433A synthesizer, resultant quantity is 0.1mmol.Protected amino acid, HOBt are Shanghai gill biochemical corp product, and DCC is from J﹠amp; K Chemical LTD., DCM, NMP are Bo Maijie company product, and all the other are domestic chromatographically pure/analytical reagent.Fmoc amino acid side chain protecting group is: trityl Trt (Cys, Asn), ethanamide methyl Acm (Cys), tertiary butyl tBu (Ser, Tyr), 9-phenyl fluorene methyl Pbf (Arg), tertbutyloxycarbonyl Boc (Lys).Resin is Wang-Fmoc-Cys (Trt) resin (Tianjin with become company).

Peptide-resin after synthetic is put in lysate (8.8ml TFA/0.5gDTT/0.5mL H ₂The O/0.2mL tri isopropyl silane) in, room temperature (25 ℃) stirs 3h, after filtration, ether sedimentation, dilute acetic acid dissolving, obtains to take off the linear peptides crude product of Side chain protective group after freeze-drying.

(2) oxidative folding (uses HPLC to separate two kinds of products, distinguishes more directly perceived with retention time with enrichment; 14.483min product may be the disulfide linkage isomer of target polypeptides))

The Bt1.3 linear peptides is at room temperature folding with air oxidation process.Folded formation is: 0.1M NH ₄HCO ₃Damping fluid, pH 8.2-8.4, peptide concentration 0.1mg/ml.Magnetic agitation 24-48h, folding process is monitored with HPLC.Folding complete, regulate pH value to 5 with dilute acetic acid, termination reaction obtains folding solution and carries out HPLC and analyze (C18 Calesil ODS-100,5 μ m, Φ 4.6 * 250mm; Flow velocity: 1ml/min; Detect wavelength: 214nm), carry out gradient elution with water (A contains 0.1%TFA (v/v)) and acetonitrile (B contains 0.1%TFA (v/v)), gradient is: 0-1min, 5%-10%B; 1-25min, 10%-50%B; 25-28min, 50%-80%B.HPLC analyzes and shows that (Fig. 2 A, A are that the folding product HPLC of Bt1.3 oxidation step analyzes, and a is linear peptides; B is folding product I of step and an II) oxidative folding mainly forms two kinds of products, i.e. I peak and II peak, retention time is respectively 14.251min and 14.483min.

To fold solution with HPLC (Waters Delta Prep 4000) enrichment (preparative column: C18, Nucleosil, 25mm * 100mm, Dalian materialization institute; Loading speed: 2.5ml/min).After end of the sample, water (containing 0.1%TFA) desalination is rinsed approximately two column volumes, flow velocity 4ml/min; Then use 90% acetonitrile (containing 0.1%TFA) wash-out desired polypeptides, flow velocity 5ml/min, 214nm detects under wavelength and collects elutriant.The elutriant of collecting is boiled off most of acetonitrile, obtain folding thick peptide after freeze-drying.

(3) purifying and analysis

Folding thick peptide carries out purifying (Kromasil C18,5 μ m with the HPLC method; Flow velocity: 3ml/min; Detect wavelength: 230nm), as gradient elution, gradient is: 0-1min, 5%-12%B with water (A contains 0.1%TFA (v/v)) and acetonitrile (B contains 0.1%TFA (v/v)); 1-25min, 12%-45%B; 25-28min, 45%-80%B.Collect target peak I peak (retention time is 12.56min), obtain polypeptide sterling Bt1.3-I after freeze-drying.Sterling is analyzed (condition is analyzed with above-mentioned Bt1.3 is folding) through HPLC, and its retention time is 14.251min (Fig. 2 A-c, c are the product I after purifying).

It is 1907.87 that mass spectrum (Pro Flex III type MALDI-TOF, Bruker company) is measured its specific charge " M+1 ", and (theoretical value is 1907.81) conforms to calculated value.

2, the determination of disulfide bond of Bt1.3

The disulfide linkage mode of connection of natural α conotoxin is mainly (C1-C3, C2-C4), therefore, the synthetic Bt1.3 that has contained the arrangement of this kind disulfide linkage, be total to sample injection method by HPLC, thus the disulfide linkage mode of connection of the folding middle first peak (being polypeptide sterling Bt1.3-I) of judgement Bt1.3 oxidation step.

(1) linear peptides is synthetic

Synthesizing linear peptide RKC (Acm) CSNPAC (Acm) NRYNPAIC.Method is with the Bt1.3 linear peptides that does not contain Cys (Acm) synthetic ((1) in 1).

(2) two-step oxidation folds and enrichment

Adopted for two steps folding, concrete steps are as follows: the first step is that atmospheric oxidation is folding, forms the polypeptide (the disulfide linkage mode of connection is 2-4) that contains a pair of disulfide linkage.Folded formation is: 0.1M Tris-HCl damping fluid, and pH 7.7, peptide concentration 0.2mg/ml, magnetic agitation is 28h approximately.Use the dilute acetic acid termination reaction after folding completing, after enrichment desalination freeze-drying, carry out second step iodine oxidative folding.Reaction conditions: 10mM iodine liquid mixes with the folding product equal-volume of the first step of 0.4mg/ml, after lucifuge reaction 10min, adds appropriate ascorbic acid solution termination reaction, and the HPLC assay products shows that this step oxidation mainly forms a kind of product.Obtain the disulfide linkage mode of connection and be " C1-C3, C2-C4 " single product III (Fig. 2 B-c) through enrichment, freeze-drying, purification step ((2) (3) of enrichment, purification process same 1) again; Fig. 2 B is that folding product III of two step of Bt1.3 reaches and folding product I (polypeptide sterling Bt1.3-I) of a step sample introduction HPLC analysis altogether, and a is linear peptides; B is the folding product of the first step; C is the folding product III of second step.)。

The determination of disulfide bond of (3) one folding products of step

A folding product B t1.3-I (polypeptide sterling Bt1.3-I) of step in above-mentioned product III and 1 is mixed sample introduction, elution requirement with 1 1) middle sample analysis condition.HPLC analyzes and shows consistent (Fig. 2 B-d of both retention time, d is the biased sample of folding product I and III, be 14.251min), illustrate that Bt1.3 folds the disulfide linkage mode of connection of product I (polypeptide sterling Bt1.3-I) identical with two folding product III of step, be " C1-C3, C2-C4 ".After gradient further reduced, both still guaranteed single elution peak.

The application of embodiment 2, Bt1.3-I polypeptide

One, analgesic activities evaluation

1, build rat sciatic nerve hemisection model

Utilize rat sciatic nerve hemisection model as the analgesic model.This model be to rat sciatic nerve directly puncture damage, can bring out strong machinery pain quick, autophagy seldom appears in animal, analog neuron source property pain preferably, the surgical procedure simple and fast, be a kind of good analgesic model simultaneously.

The SD rat, male, body weight 200-220g provides (the SCXK-2007-004 of army) by Test Animal Centre, Academy of Military Medical Sciences, P.L.A.Reference literature (Seltzer et al.Pain .1990,43, method 205-218.) builds sciatic nerve hemisection rat model.Concrete steps are as follows: rat exposes right sciatic nerves in a thigh section high position under anesthesia and aseptic technique, under 25 times of magnifying glasses, carefully neural dorsal part and surrounding tissue are separated to the far-end of PB and semitendinosus bifurcation in sciatic nerve-trunk, clamp neural dorsal part with little mosquito forceps, a 6-0 silicon scrap prodn. line is sewed up in nerve trunk, neural dorsal part 3/1-2/1 is received in the line cover, then tightens the line cover.Muscle and skin are all sewed up with cotton thread.

Perform the operation and use Ugo 37215 type tenderness instrument (Ugo Basile after seven days, Italy), test Pain Regulation In The Rat value, pressure pain point is positioned at the interphalangeal joint place of rat right hind leg the one or two toe, with compare before art, the threshold of pain 50% above person that descends is considered as the successful rat of modeling.

2, utilize the analgesic activities of rat sciatic nerve hemisection model determination Bt1.3

The rat of modeling success is divided into 5 groups at random, 8 every group.First group of intramuscular injection physiological saline (0.2ml) is as negative control, second group of intramuscular injection 0.2ml physiological saline contains 0.3nmol Vc1.1, and (Vc1.1 is synthetic by this laboratory, sequence is GCCSDPRCNYDHPEIC*) as positive control, the 3rd group to the 5th group rat intramuscular injection 0.2ml physiological saline respectively contains the alpha-conotoxin Bt1.3-I that 0.06nmol, 0.15nmol and 0.3nmol above-described embodiment 1 obtain.After administration 1.5h, the threshold of pain of test rat changes.Experiment arranges three repetitions, and threshold of pain is got the mean value of three repeated experiments of 8 rats.

Test result shows, before administration, the Pain Regulation In The Rat mean value of negative control group is 127 grams, positive controls threshold of pain mean value is 101 grams, the Pain Regulation In The Rat mean value of experimental group is respectively 105 grams, and (0.06nmol/ only organizes, the 3rd group), 96.9 grams (0.15nmol/ only organizes, the 4th group) and 99.4 grams (0.3nmol/ only organizes, the 5th group).

After administration 1.5h, the threshold of pain mean value of positive control is 119 grams, and the threshold of pain mean value of physiological saline (negative control group) is 129 grams; 3 groups of experimental group: 0.06nmol/ only organize (the 3rd group), 0.15nmol/ and only organize the threshold of pain that (the 4th group), 0.3nmol/ only organize (the 5th group) and be respectively 115.63 grams, 112.5 grams and 125.63 grams;

As can be seen from the above, after administration 1.5h, the analgesic effect of Bt1.3-I presents dose-dependently, and along with dosage increases, the corresponding raising of Pain Regulation In The Rat value is respectively 10.25%, 16.11% and 26.70%, and analgesic effect is obvious, and Vc1.1 is suitable with positive control.Each experimental group Pain Regulation In The Rat value increase rate is seen Fig. 3, histogram graph representation PNL rat muscle injecting normal saline, Vc1.1 and various dose Bt1.3, the average threshold of pain increase rate (%) after 1.5h.Analgesic effect presents dose-dependently.

Two, the effect of Bt1.3-I to the different subunits of Neuronal nicotinic acetylcholine receptor

1, the separation of xenopus leavis oocytes and cultivation

(U.S. Enasco company LM00535MX) in anesthesia on ice, takes out approximately 1cm of 3-4 leaf from belly with Xenopus laevis ³The ovum leaf, be placed on OR-2 nutrient solution (82mM NaCl, 2.5mM KCl, 1.0mM MgCl ₂6H ₂O, 5.0mM HEPES) after repeatedly cleaning in, add collagenase solution (Sigma, lot number: 020M1225V) (1-2mg/ml), 65rpm effect 1 hour.Then the Digestive system that inclines cleans 5-6 time with OR-2 solution, changes ND-96 solution (96mM NaCl, 2.0mM KCl, 1.8mM CaCl over to ₂, 1.0mM MgCl ₂.6H2O, 5mM HEPES, pH7.4).Select V, VI phase mature oocyte in sharp contrast, that external form is round, again be placed in fresh ND-96 nutrient solution, 18.2 ℃ save backup.

2, the expression of subunit

Coding rat nervous system type junctional acetylcholine receptor subunits added cap cRNAs (α 3 (GI:148747113), β 2 (GI:9506484), β 4 (GI:148747284, extract rat cerebral tissue, use primer by sequence, acquisitions cloned, transcribed in amplification) mixing is dissolved in 50.6nl and goes (mol ratio 1: 1 in RNA enzyme water, every kind of subunit 15-20ng), then all be injected into above-mentioned xenopus leavis oocytes.The ovocyte that injection is good is placed in ND-96 solution, and after 18.2 ℃ of cultivation 48h, electric current expressed in record.

3, electrophysiological recording

Conventional two electrodes voltage clamp recording method is adopted in experiment.(sutter company, all with the perfusion of 3mol/L KCl solution, resistance is 0.5-1.5M Ω for P-97) drawn glass microelectrode, voltage electrode and galvanic electrode, clamps down on voltage and is-70mV to use microelectrode to draw instrument.In experiment with the ovocyte (contain the ovocyte of α 3 β 2 or α 3 β 4 with vagusstoff solution perfusion after, have obvious electric current to be excited, prove its successful expression α 3 β 2 or α 3 β 4) of express alpha 3 β 2 or α 3 β 4.Insert the bottom and be stained with in the culture dish of grid, first with contain 200 μ M vagusstoffs (Sigma company, lot number: ND-96 solution perfusion 137751132408192), flow velocity 3ml/min is to excite the vagusstoff gating current of subunit; Then continous perfusion after the Bt1.3-I that obtains in embodiment 1 being mixed with finite concentration acts on ovocyte.Use Axoclamp 900A type amplifier (Axon Instruments Inc., Union City, CA) and pClamp 10.0 software collections and analytical data, graphicprocessing adopts Origin 8.0 softwares.Each mass action 4-5 piece ovum of Bt1.3, its inhibition to the vagusstoff excitation current represents with average inhibiting rate ± standard deviation; Take the solution that do not add Bt1.3 as contrast.

It is 4 described that electrophysiology the results are shown in Figure, and wherein, a)-c) is respectively 10nM, 50nM and 1000nM Bt1.3 (with the inhibition of Bt1.3-I to rat α 3 beta 2 subunit type vagusstoffs (200 μ M) excitation current; D) be 1000nM Bt1.3 to the inhibition of rat α 3 β 4 hypotype ACh excitation currents, 10nM, 50nM and 1000nM Bt1.3 can suppress respectively 11.5%, 33.8% and 65.3% neurons of rats type α 3 beta 2 subunit type vagusstoff excitation currents.And 1000nMBt1.3 is faint to neurons of rats type α 3 β 4 hypotype effects.

Claims (6)

1. a peptide species, its aminoacid sequence is the sequence 2 in sequence table.

2. the encoding gene of the described polypeptide of claim 1.

3. encoding gene according to claim 2, it is characterized in that: the encoding gene of described polypeptide is the DNA molecular shown in sequence 1 in sequence table.

4. polypeptide according to claim 1 is characterized in that:

The mode of connection of the disulfide linkage of described polypeptide is Cys1-Cys3 and Cys2-Cys4; The Cys that begins from the aminoterminal of described polypeptide is denoted as respectively Cys1, Cys2, Cys3, Cys4 in order.

5. polypeptide claimed in claim 1 has application in the product of analgesia function in preparation.

6. application according to claim 5 is characterized in that: described product is medicine.

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