CN102604960B - Neurotoxin S10a of South China Sea Conus striatus, coding sequence and application of the neurotoxin - Google Patents

Abstract

The invention provides a Chinese South China Sea conotoxin gene S10.1 and its encoded polypeptide S10a, as well as the application of the polypeptide S10a in the preparation of a tool drug and analgesic drug for neurobiological research. The present invention adopts the method of constructing a cDNA library, clones the conotoxin gene S10.1 obtained from the conotoxin gene S10.1 obtained by cloning from the venomous tube of the linen cone snail in the South China Sea, analyzes the base sequence through the tool software SEQTOOL, and determines the above-mentioned conotoxin gene S10.1 The amino acid sequence of the precursor peptide of the encoded polypeptide S10a is shown as sequence 2 in the sequence listing. The polypeptide S10a provided by the invention has good analgesic effect, and can be used to prepare tool medicine and analgesic medicine for neurobiology research.

Description

South China Sea strain line conus neurotoxin S10a and encoding sequence and purposes

Technical field

The present invention relates to a peptide species and encoding sequence thereof and application, relate in particular to a kind of strain line conotoxin polypeptide and encoding sequence and application.Belong to biological field.

Background technology

Cone shell (Cone snails) belongs to Mollusca (Mollusca), Gastropoda (Gastropoda), Caenogastropoda (Neogastropoda), Conidae (Conidae), Conus (Conus) on the taxonomy.According to estimates, existing cone shell has kind more than 700 approximately, is distributed widely in each torrid zone and marine site, subtropics in the whole world, and is wherein maximum with the Indian Ocean-Pacific region.Cone shell perches the karang in warm temperate zone and tropical marine site more, the shoal, and tide soaks zones such as area, and minority is perched in the ballow of several meters of depth of waters rice surplus 200, and daytime or ebb back are perched under marine alga or in the coral hole, go out to look for food the spring and summer breeding night.The external form of cone shell is easy to identification, and breechblock is back taper, and extremely solid has long ditch shape faucal.The cone shell shell is heavy or light, and color and decorative pattern are bright-colored colorful, and cross band, thin spot, mottled zone etc. are arranged.The shell top of some cone shell is flat, and other cone shells then have a spiral shell tower portion of stretching out.

Nearly 30 years studies show that, every kind of cone shell individuality contains 50-200 kind venom composition, and with its called after conotoxin peptide.Conotoxin peptide is the very short and small peptide molecule of a class, generally contains 7-40 amino acid, will lack than the polypeptide toxin (40-80 amino acid) of species such as snake, scorpion, spider and sea anemone.Conotoxin peptide is by the term single gene direct coding, and conotoxin peptide is called precursor peptide (prepropeptide) from the fragment that mRNA translates 50-120 amino-acid residue.Precursor peptide is made of three parts, the i.e. signal peptide (signal peptide) of the high conservative of forming near 19-27 amino acid of N-end, the mature peptide (mature peptide) that the height of the comparatively conservative leading peptide (pro-region) that 20-40 middle amino acid is formed and 7-40 amino acid composition of C-end makes a variation.

Conotoxin peptide is done as a whole, has diversity action target spot beyond imagination.Up to the present, the neuronal target of the conotoxin peptide effect of having illustrated mainly is divided into the ionic channel acceptor, comprises Na ⁺, Ca ²⁺, K ⁺Passage and neurotransmitter receptor comprise acetylcholine receptor, 5-hydroxytryptamine receptor (5-HT ₃R), nmda receptor (N-methyl-D-aspartateReceptor), norepinephrine associated receptor (NA transporter, α 1-adrenoceptor) etc.Single individuality from conotoxin peptide then has the characteristics of site-specific nature, the various acceptors of the specific differentiation of energy, even the hypotype of various acceptors, and this makes the valuable source storehouse of its effective tool that becomes neurobiology research and new drug development.The narrow spectrum valtage-gated calcium channel of N-type that acts on of ω-MVIIA, its intrathecal injection have more than morphine to be renderd a service and needn't worry tolerance and the habituation of medicine, therefore is used for the treatment of severe chronic pain by drugs approved by FDA in 2004.Derive from other omega-conotoxins of different cone shell species, although their aminoacid sequence differences, but difference structurally is not very big, as GVIA, GVIC, GVIIA and the GVIIB etc. from ground-tint cone shell (C.geographus), with from unreal cone shell MVIIA, MVIIC and from the omega-conotoxins such as SO3 of strain line cone shell (C.striatus), similar effect is arranged aspect pharmacology.

China has found to have approximately more than 80 kind of cone shell now, mainly is distributed in the Nansha Islands, the Xisha Islands, Hainan Island and surrounding waters, Taiwan, and minority is distributed in Guangdong, ALONG GUANGXI COAST.The toxinology research of most of cone shell species does not also launch.Because different conotoxins has its specific pharmacotoxicological effect scope and biologic activity, thereby make conotoxin have important theoretical research value and using value.Multifarious conotoxin both can be used as the lead compound of design new drug (being used for the treatment of great difficult and complicated cases) again directly as natural drug.Further investigation conotoxin and physiologic function thereof, not only significant to the ocean pharmacy industry, and can be the exploitation protection of China's marine conidae species resource and utilize the most important theories foundation is provided.

Summary of the invention

The object of the present invention is to provide a kind of South China Sea strain line conotoxin gene S10.1.

Another purpose of the present invention provides the conotoxin polypeptide S10a of above-mentioned conotoxin gene S10.1 coding.

Still a further object of the present invention provides above-mentioned conotoxin polypeptide S10a in the instrument medicine of preparation neurobiology research and the application in the analgesic.

The selected strain line cone shell of the present invention (Conus striatus) is picked up from Sanya, Hainan Province.

The present invention adopts the method in construction cDNA library, and the clone obtains conotoxin gene S10.1 from South China Sea strain line cone shell poison pipe.

The structure in South China Sea strain line cone shell poison pipe cDNA library: separate the poison pipe, extract the synthetic cDNA first chain product of total RNA reverse transcription; Signal peptide and 3 ' UTR with the T superfamily is primer again, is template with cDNA one chain, carries out gene clone by PCR, and the PCR product is linked to each other with pGEM-T Easy Vector, after connecting product and transforming, is coated with flat board respectively, and the picking mono-clonal checks order from the flat board.Obtain South China Sea strain line conotoxin gene S10.1, its dna sequence dna is nucleotide sequence shown in sequence in the sequence table 1.

Utilize tool software SEQTOOL that the base sequence of above-mentioned conotoxin gene S10.1 is analyzed, obtain its maximum reading frame, length 189bp, the conotoxin precursor peptide of 62 amino-acid residues of codified.By further analysis, determine the precursor peptide of the toxin polypeptide S10a that conotoxin gene S10.1 encodes and mature peptide aminoacid sequence aminoacid sequence shown in sequence in the sequence table 2,3 of supposition: the mature peptide of above-mentioned toxin polypeptide S10a is made up of 13 amino acid, theoretical molecular is 1429.54 dalton, feature with typical conotoxin polypeptide primary structure, intramolecularly has two pairs of disulfide linkage, first becomes a pair of disulfide linkage with the 4th halfcystine, and second becomes another to disulfide linkage with the 3rd halfcystine.

The present invention utilizes the solid state chemistry synthesis method to synthesize above-mentioned toxin polypeptide S10a.Adopt the instrument synthesis strategy, use the FmocPHOBtPDCC method, Rink resin and Fmoc-amino acid, coupling agent are DCC-HOBT, and piperidines takes off the Fmoc-base, and the synthetic handbook of synthesis step reference instrument carries out.Peptide-resin cracking reagent set one-tenth and peptide recovery method are identical with manual synthetic method.By solid state chemistry synthesizing linear polypeptide, crossed loops changes into the mature peptide with two pairs of disulfide linkage and C-terminal amidation modification then, identify that through HPLC reaching 99%, MALDI-TOF by the synthetic neurotoxin polypeptide S10a purity of solid state chemistry synthesis method identifies that its molecular weight is correct.

In the central analgesia effect experiment of conotoxin polypeptide S10a provided by the invention on mouse hot plate method mensuration physiological models, show tangible central analgesia effect, and drug effect is better than the positive control drug pethidine hydrochloride.Therefore, toxin polypeptide S10a provided by the invention can be applicable to prepare instrument medicine and the analgesic of neurobiology research.

Description of drawings

Fig. 1 South China Sea strain line cone shell poison pipe gene S10.1PCR amplification gel electrophoresis figure.

The mature peptide of Fig. 2 South China Sea strain line conotoxin gene S10.1 coding and the comparative result of T superfamily (skeleton X) conotoxin mature peptide;

Fig. 3 connection carrier pGEM-T Easy Vector annular collection of illustrative plates and relevant sequence site;

The HPLC analysis chart of the synthetic conotoxin polypeptide S10a of Fig. 4 solid state chemistry;

Fig. 5 identifies the MALDI-TOF mass spectrum of the synthetic conotoxin polypeptide S10a molecular weight of solid state chemistry;

Fig. 6 mtt assay is measured the cell survival rate of conotoxin polypeptide S10a influences interpretation of result figure;

The analgesia drug effect experimental result picture of Fig. 7 conotoxin polypeptide S10a on the mouse physiological models.

Embodiment

Below in conjunction with specific embodiment, further specify technical scheme of the present invention.Should be understood that following examples only are used for the present invention being described and not limiting the present invention in any form.

Embodiment 1: the extraction of the total RNA of strain line poison tubing and toxin cDNA clone

The extraction of total RNA is with reference to Gibcol BRL company

LS reagent specification sheets carries out.LA Taq archaeal dna polymerase, 10 * PCR Buffer, DNA ladder buy from TaKaRa company; DNTP mix is available from Promega company; The PCR primer is synthetic by Invitrogen company; Other organic reagents are homemade analytical pure, available from Guangzhou Chemical Reagent Factory.

Get breechblock alive, with kevel fragmentation spiral shell shell, expose spiral shell meat, careful also sharp separation poison tubing is put into liquid nitrogen and abundant the grinding rapidly after weighing on ice, 15 times of bulking values of adding

LS reagent (being to add 15ml in the 1g tissue), fully homogenate in the ice bath is preserved in-80 ℃ of refrigerators in order to extracting total RNA.Get the 50-100mg tissue sample, use 0.75ml

The homogenate of LS reagent, 15-30 ℃ leaves standstill 5min.Add the 0.2mL chloroform then, thermal agitation is after 15 seconds, and 15-30 ℃ leaves standstill 2-15min.4 ℃, 12, the centrifugal 15min of 000rpm gets the upper strata water.Add the 0.5ml Virahol, 15-30 ℃ leave standstill 10min after, 4 ℃, 12, the centrifugal 10min of 000rpm abandons supernatant.Add 1ml 75% ethanol rinsing precipitation again, the centrifugal 5min of 5,000rpm abandons supernatant, and the ethanol of trace in the sample is removed in vacuum-drying, adds the deionized water of an amount of no RNase.Get 1 μ l and carry out 1% agarose gel electrophoresis, 1 μ l is with concentration and the purity of ultraviolet spectrophotometer estimation RNA, and-20 ℃ of preservations are standby.

1) cDNA first chain is synthetic

The synthetic SMART that uses Clontech company of cDNA first chain ^TMPCR cDNA Synthesis Kit tests to specifications.In 5 μ l reaction systems, add following component (in operation on ice): the total RNA of 1 μ g spiral shell poison pipe, each 1 μ l of SMARTIII olignucleotide and CDS III/3 ' PCR primer, with ultrapure water polishing volume, mixing, of short duration centrifugal.72 ℃ of incubation 2min, ice bath 2min, of short duration centrifugal, make mixture combine in the pipe end.Flick tube wall after adding the dNTP Mix of DTT, 1 μ l 10mmol/L of 2 μ l, 5 * First Strand Buffer, 1 μ l 20mmol/L and 1 μ l PowerScript RT more successively, mixing is also of short duration centrifugal.Put synthetic first chain of 42 ℃ of reactions of PCR instrument 1hr reverse transcription, ice bath termination reaction ,-40 ℃ of preservations.

2) cDNA of T-superfamily conotoxin clone

The PCR primer sequence:

Upstream primer 5 ' ATGCGCTGYYTCCCAGTCTT 3 '

Downstream primer 5 ' AACAACACGCTGCCACTTGC 3 '

PCR system and PCR program are as shown in table 1 below:

Table 1

The PCR system		The PCR program
				Component	Volume (μ l)	95 ℃ of pre-sex change	3min
CDNA first chain	1	30 circulations
				Primer 1 (10 μ M)	1	95℃	45sec
Primer 2 (10 μ M)	1	58℃	45sec
				DNTP mixture (10Mm)	0.4	72℃	60sec
The T4Taq polymerase	0.5	72 ℃ of extensions	10min
				10 * PCR reaction buffer	2
The deionization tri-distilled water	14.6
				Cumulative volume	20

Get 5ul PCR product and detect amplification with 1.8% agarose gel electrophoresis, then prepare the above-mentioned reaction system of 50ul if any specific band, amplify by above-mentioned reaction conditions.

3) purifying of PCR product

With step 2) the PCR reaction product that obtains carries out purifying, voltage 120V electrophoresis 25 minutes with 1.8% gel.Fig. 1 is the gel electrophoresis figure of South China Sea strain line conotoxin gene S10.1PCR amplification, downcuts the sepharose piece that contains target nucleic acid band (about 300bp), presses the GEL EXTRACTION KIT specification sheets operation of OMEGA BIOTEK company.The sepharose piece is weighed, press 1g/ml and convert, add the long-pending Binding Buffer damping fluid of 4-5 times of colloid, in 55-60 ℃ of dissolving 7min, up to melt and dissolved fully, coagulant liquid is joined HiBind ^TMOn the DNA post (can in conjunction with the DNA of 25 μ g), can add 800 μ l samples (can repeat to add) at every turn, the centrifugal 1min of 10,000g, the other working sample of degree score is measured 260nm, 280nm wavelength absorption value at spectrophotometer, tentatively abandons waste liquid; Wash post once with 750 μ l through the DNA of alcohol dilution wash buffer, the centrifugal 1min of 10,000g abandons liquid, repeats to wash post once with DNA wash buffer again; HiBind with sky ^TMThe DNA post is abandoned trace solution at the centrifugal 1min of 10,000g; Wash post with 30 μ l sterilization deionized water or TE damping fluid, the centrifugal 1min of 10,000rpm collects DNA, and dna solution is-20 ℃ of preservations.Production concentration identify is reclaimed in 1.8% gel electrophoresis, obtains the PCR product behind the purifying.

4) the PCR product cloning is to the T-carrier

By DNA Ligation Kit explanation the purpose fragment in the PCR product behind the purifying is connected into pGEM-T Easy Vector.

Linked system is as shown in table 2 below:

Table 2

Component	Volume (μ l)
		The purpose segment	4
pGEM-T Vector	0.5
		2×Rapid Ligation Buffer	5
T4 DNA Ligase	0.5
		Cumulative volume	10

4 ℃ of connections are spent the night.

5) connecting product transforms

To connect product transformed into escherichia coli DH5 α bacterial strain.

At first use CaCl ₂Legal system is equipped with the bacillus coli DH 5 alpha competent cell.Picking bacillus coli DH 5 alpha list colony inoculation is not in containing antibiotic LB liquid nutrient medium, 37 ℃ of shaking culture 16-18 hour activation bacterial strains, being inoculated in 50ml with 1: 100 volume does not then contain in the LB liquid nutrient medium of acillin, 37 ℃, 250rpm shaking culture are about 2-3 hour, at OD ₆₀₀During=0.3 left and right sides, with bacterium liquid ice bath 15 minutes, centrifugal 5 minutes of 4 ℃ of 4000rpm were inverted and remove most supernatant, add the 100mM CaCl of the precooling that is equivalent to stock culture 1/2 volume ₂Resuspended precipitation, ice bath 10 minutes, 4 ℃, centrifugal 5 minutes of 5,000rpm abandons supernatant, again with the 100mM CaCl of the precooling that is equivalent to stock culture volume 1/25 ₂Resuspended precipitation.Every pipe packing 200 μ l, it is available after 2 hours to put 4 ℃ of refrigerators, uses transformation efficiency constant in 48 hours.

Get the competent cell of three pipe prepared fresh, each adds vector plasmid DNA, connects product or does not add any DNA, respectively behind the mixing, ice bath 30 minutes, 42 ℃ of thermal shocks 90 seconds, ice bath 2 minutes adds the LB liquid nutrient medium of 200 μ l Amp-, 37 ℃ of gentle jolting recovery cells 30 minutes are got an amount of converted product and are coated Amp ⁺The LB flat board on, 37 ℃ of inversion incubators were cultivated 16 hours, observed the colony growth situation.

6) bacterium colony PCR preliminary evaluation positive colony

Get 20 sterilization 1.5ml tubules, add 20 μ l LB substratum, 20 positive colony branches of picking are clipped in each tubule in the super clean bench of step 5) cultivation bacterium colony.

PCR system and PCR program are as shown in table 3 below:

Table 3

The PCR system		The PCR program
				Component	Volume (μ l)	95 ℃ of pre-sex change	3min
As above preparation contains bacterium culture medium	1	30 circulations
				F(10μM)	1	95℃	45sec
R(10μM)	1	60℃	45sec
				DNTP mixture (10 μ M)	0.4	72℃	60sec
The T4Taq polymerase	0.5	72 ℃ of extensions	10min
				10 * PCR reaction buffer	2
The deionization tri-distilled water	14.6
				Cumulative volume	20

7) enlarged culturing of positive bacterium colony and plasmid extract

The bacterium liquid of getting the bacterium colony PCR positive is added to containing in the Amp LB substratum of 5ml, and 37 ℃ of incubated overnight are extracted plasmid with OMEGAmini plasmid extraction kit.Picking positive colony list bacterium colony, 37 ℃ of incubated overnight 14-16hr; Get 5ml bacterium liquid, the centrifugal 1min of 10,000g; Abandon supernatant, add 250 μ l solution I/RNase, re-suspended cell; Add 250 μ l solution II, put upside down soft mixing 4-6 time, obtain clear soln, room temperature is placed slightly; Add 350 μ l solution III, mixing is up to white precipitate occurring; Behind the centrifugal 10min of 10,000g, supernatant is transferred to the HiBind that places on the 2ml centrifuge tube sleeve pipe ^TMOn the DNA post; The centrifugal 1min of 10,000g abandons liquid; Wash post with 500 μ l HB buffer, the centrifugal 1min of 10,000g abandons liquid; Wash post once with 750 μ l through the DNA of alcohol dilution wash buffer, the centrifugal 1min of 10,000g abandons liquid, repeats to wash post once; Empty HiBind ^TMDNA post again 10, the centrifugal 1min of 000g abandons trace liquid; 40 μ l sterilization deionization washing post twice, 10, the centrifugal 1min of 000g, dissolving plasmid.

8) contain the T vector plasmid order-checking of inserting segment

Adopt ABI PRISM 3700 automatic sequencers of Perkin Elmer company to carry out examining order, sequencing reaction is pressed ABIPRISM BigDye ^TMThe specification sheets of Terminator Cycle Sequencing Ready Reaction Kits is operated, and uses T7 and SP6 primer to carry out two-way sequencing.

Embodiment 2: mensuration and the analysis of conotoxin gene S10.1 sequence

Cloning and sequencing obtains conotoxin gene S10.1 from the South China Sea strain line cone shell poison pipe cDNA library that embodiment 1 makes up.Conotoxin gene S10.1 belongs to cance high-expression gene, utilizes tool software SEQTOOL that its base sequence is analyzed, and obtains its maximum reading frame, length 189bp, the precursor peptide of 62 amino-acid residues of coding.Further analyze and show that the 1-22 amino acids residue of this precursor peptide is its signal peptide zone, its 49th residue is arginine, can think standard protein hydrolysis signal, and leading peptide and the mature peptide of precursor peptide partly separated.So determine that conotoxin gene S10.1 mature peptide sequence is QTCCGYRMCIPCG ^*( ^*The expression amidation is modified), molecular weight is 1446.4 dalton, and thinks skeleton X toxin the most similar (as shown in Figure 2) in mature peptide and the T superfamily of above-mentioned toxin gene S10.1 coding.

Embodiment 3: the solid state chemistry of polypeptide S10a is synthetic

The synthetic neurotoxin S10a sequence of solid state chemistry is QTCCGYRMCIPCG ^*( ^*The expression amidation is modified), molecular weight is 1446.4 dalton, and first becomes a pair of disulfide linkage with the 4th halfcystine intramolecularly, and second becomes another to disulfide linkage with the 3rd halfcystine.And the 11st amino-acid residue proline(Pro) P replaces with 4-Hydroxyproline O in building-up process.Adopt the instrument synthesis strategy, use the FmocPHOBtPDCC method, Rink resin and Fmoc-amino acid, coupling agent are DCC-HOBT, and piperidines takes off the Fmoc-base, and the synthetic handbook of synthesis step reference instrument carries out.Peptide-resin cracking reagent set one-tenth and peptide recovery method are identical with manual synthetic method.As shown in Figure 4, its purity of HPLC Analysis and Identification of the synthetic conotoxin polypeptide S10a of solid state chemistry is 99.06%; As shown in Figure 5, MALDI-TOF identifies that the molecular weight of conotoxin polypeptide S10a is correct.

Embodiment 4: the cytotoxicity of strain line conotoxin polypeptide S10a is identified

General planning: mtt assay

Experimental procedure: with MDCK (Madin-Darby canine kidney(cell line)), H9C2 (rat myocardial cell), chang ' s liver (CCL 13), L929 (l cell) is with about 10 ⁵The density of individual/ml is inoculated in 96 orifice plates, and 100 μ l are inoculated in every hole, put in the CO2 incubator and are cultured to logarithmic phase.By default concentration gradient, adding is respectively 100 μ M, 10 μ M, the S10a of 1 μ M, at least 5 repetitions of each gradient then.Control group adds the solvent that isopyknic sample dissolution is used.Continue to cultivate after 6 hours, every hole adds the MTT (5mg/ml) of 20 μ l, places 37 ℃ of incubations then 4 hours.After carefully removing supernatant, every hole adds the DMSO of 100 μ l, and about 10min dissolution precipitation that vibrates detects OD value, wavelength 570nm with microplate reader subsequently.Obtain cell survival rate under each sample concentration: the average OD value of the average OD value/control group of survival rate %=test group * 100% with following formula.

Interpretation of result: can be got by experimental result, when middle and high concentration, the L929 of S10a, H9C2, MDCK are when 3h, and beginning has certain lethal effect; When 6h, the effect of toxin reaches the highest.And when lower concentration, the L929 of S10a still has certain toxic action; Growth to H9C2 and MDCK does not then have influence substantially.Under high, medium and low three concentration, the chang ' s of S10a liver does not have toxic action substantially.Illustrate that S10a has different toxic actions to dissimilar cells, and along with activity successively decreases, toxic action reduces (see figure 8).

Embodiment 5: the central analgesia effect experiment on the physiological models

General planning: mouse hot plate method

Choose Healthy female SPF level kunming mice, body weight 20 ± 2g is available from Guangdong university of TCM Experimental Animal Center.Mouse is put on the GJ-8402 type hot plate analgesia instrument (Chengdu Tai Meng science and technology limited Company) that is heated to 55 ℃ in advance, is the threshold of pain of this mouse with stopwatch record mouse from dropping into hot plate to the time that metapedes or jump occur licking (second).Measuring the threshold of pain of every mouse before the abdominal injection earlier (surveys twice altogether, is no more than 20 seconds persons for qualified with mean value.) injection back 0.5h, 1h, 2h, 3h, 4h measures once, surpasses 60 seconds as threshold of pain, namely stops test and by calculating (time is of a specified duration excessively, easily scalds foot) in 60 seconds.Experiment finishes, and improves percentage by the mean value calculation threshold of pain of being surveyed, the threshold of pain, that is:

Percentage=(the preceding average threshold of pain of average threshold of pain-medication after the medication)/preceding mean value of medication is improved in the threshold of pain.

Experimental design: choose Healthy female SPF level kunming mice, body weight 20 ± 2g is divided into 5 groups at random, 10 every group, each one group of negative control group, positive controls and the high, medium and low test group of conotoxin polypeptide S10a is set.Sheath inner injecting medicine-feeding, every mouse 10ul.Specific as follows:

First group: negative control group, 10 mouse are all injected 10ul physiological saline for every;

Second group: positive controls, 10 mouse are all injected 10ul Pethidine (1.25mg/kg) for every;

The 3rd group: S10a sample high dose group, 10 mouse are all injected 10ul S10a (0.7mg/kg) for every;

The 4th group: dosage group in the S10a sample, 10 mouse are all injected 10ul S10a (0.07mg/kg) for every;

The 5th group: S10a sample low dose group, 10 mouse are all injected 10ul S10a (0.007mg/kg) for every;

Threshold of pain after the basic, normal, high dosage group mouse administration of given the test agent S10a has raising in various degree, and data have significant difference (P＜0.01).Dosage is more high, and analgesic effect is more good.The result as shown in Figure 7, with respect to the positive control Pethidine, the onset of S10a analgesic activity is slow slightly, but long action time begins onset when 1h, reach the best use of when 3h, high dose group still has good analgesic activity during 4h.The drug effect of the middle and high dosage group of S10a all will obviously be better than the positive control drug Pethidine, is expected to for the preparation of new efficient central analgesia medicine.

Claims (4)

1. A Chinese South China Sea conotoxin gene, characterized in that the sequence is the nucleotide sequence shown in sequence 1 in the sequence listing. 2. A polypeptide encoded by the conotoxin gene according to claim 1, characterized in that: the precursor peptide sequence of the polypeptide is the amino acid sequence shown in Sequence 2 in the sequence listing. 3. The mature peptide of the polypeptide encoded by the conotoxin gene according to claim 2, characterized in that: the sequence of the mature peptide is the amino acid sequence shown in sequence 3 in the sequence listing. 4. The use of the polypeptide according to claim 3 in the preparation of analgesic drugs.

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