αO-superfamily conotoxin peptide, pharmaceutical composition and use thereof

Abstract

The present invention pertains to fields of biochemistry and molecular biology, relates to an αO-superfamily conotoxin peptide, pharmaceutical composition thereof, preparation method and use thereof. The present invention further relates to a propeptide of the conotoxin peptide, nucleic acid construct thereof, expression vector and transformed cell thereof, and fusion protein thereof. The present invention further relates to a method for blocking acetylcholine receptors as well as a use of the conotoxin peptide in the manufacture of a medicament. The new αO-superfamily conotoxin peptide of the present invention is capable of specifically blocking acetylcholine receptor (nAChRs) (e.g., α9α10 nAChR), and NMDA receptor (e.g., NR2C NMDAR), and has activity for treatment of neuralgia, addiction, and activity for treatment of chemotherapy of cancers, breast cancer, lung cancer, wound healing, epilepsia, ischemia, and thus is promising in the manufacture of analgesic, a medicament for treatment of addiction, and a tool drug for neuroscience.

Claims

1. A polypeptide, which consists of or comprises one or more same or different amino acid sequences as shown by any one of SEQ ID NOs: 10-12.

2. A polypeptide according to claim 1, wherein the polypeptide consists of or comprises SEQ ID NO: 12, wherein from the N-terminus of SEQ ID NO:12, the 1.sup.st cysteine and the 2.sup.nd cysteine form a disulfide bond, and the 3.sup.rd cysteine and the 4.sup.th cysteine form a disulfide bond; or the 1.sup.st cysteine and the 3.sup.rd cysteine form a disulfide bond, and the 2.sup.nd cysteine and the 4.sup.th cysteine form a disulfide bond; or the 1.sup.st cysteine and the 4.sup.th cysteine form a disulfide bond, and the 2.sup.nd cysteine and the 3.sup.rd cysteine form a disulfide bond; and the carboxyl terminus of the polypeptide is a free C-terminus, or amidated.

3. A fusion protein, which comprises a polypeptide according to claim 1.

4. A pharmaceutical composition, which comprises a polypeptide according to claim 1.

5. A method for preparing a polypeptide according to claim 1, comprising the following steps: 1) synthesizing a linear polypeptide by ABI Prism 433a polypeptide synthesizer or by a manual method, in which side-chain protecting groups of Fmoc amino acid are: Pmc (Arg), Trt (Cys), But (Thr, Ser, Tyr), OBut (Asp), Boc (Lys); cysteine is protected with Trt or Acm protecting group, disulfide bonds are respectively formed in a site-directed manner between corresponding cysteines; 2) cutting the linear polypeptide of step 1) from resin, using ice-ether to precipitate and wash and recover a crude product of the linear polypeptide, and using a preparative reversed phase HPLC C18 column for purification; and 3) subjecting the product obtained in step 2) to two-step oxidative folding.

6. A method for treatment and/or prophylaxis of neuralgia, breast cancer, or lung cancer a method for killing a pest, or a method for analgesia, comprising the step of administering an effective amount of a polypeptide according to claim 1.

7. The pharmaceutical composition according to claim 4, which further comprises a pharmaceutically acceptable carrier or an excipient.

8. The method according to claim 6, wherein said pest is Spodoptera Frugiperda.

9. The method according to claim 6, wherein said neuralgia is caused by a factor selected from: cancers and chemotherapy of cancers, alcoholism, ischioneuralgia, diabetes mellitus, prosopalgia, sclerosis, herpes zoster, mechanical injury and surgical injury, AIDS, head nerve paralysis, drug poisoning, industrial pollution poisoning, lymphatic neuralgia, myeloma, multipoint motor neuralgia, chronic congenital esthesioneurosis, acute spontaneous neuralgia, squeezing neuralgia, angiitis, vasculitis, ischemia, uremia, children biliary liver disease, chronic respiratory disorder, complex neuralgia, multiple organ failure, sepsis/pyaemia, hepatitis, porphyria, avitaminosis, chronic liver diseases, primary biliary cirrhosis, hyperlipidemia, leprosy, Lyme arthritis, sensory perineuritis, and allergies.

10. The method according to claim 6, wherein said treatment is adjuvant treatment.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Notation: when sources of various nAChRs and NMDA receptor subtypes are not given in the drawings, they are all corresponding rat receptors, and the sources of rat receptor types are omitted in the legends and drawings.

(2) FIG. 1: shows αO-GeXIVA mature peptide sequence (SEQ ID NO: 1) and isomers having 3 possible disulfide bond linkage ways, in which GeXIVA12 has disulfide bond linkage ways of I-II, III-IV; GeXIVA13 has disulfide bond linkage ways of I-III, II-IV; GeXIVA14 has disulfide bond linkage ways of I-IV, II-III.

(3) FIG. 2: shows in A the effects of 33 nM αO-GeXIVA12 on electric current of α9α10 nAChR. In diagram A, “C” refers to a control electric current, the arrow indicates the current track (˜0 nA) formed by the first Ach pulse with Ach pulse time of 1 s after 33 nM αO-GeXIVA12 was incubated for 5 minutes; ordinate refers to current strength in unit of nA, abscissa refers to cumulative time in unit of ms, the interval time between any of 2 adjacent current tracks is 60 s before and after incubation. 33 nMαO-GeXIVA12 totally blocks α9α10 nAChR current, and elution is very rapid. In the figure, B, C, D separately refers to concentration dose-response curves of 3 isomers, αO-GeXIVA12, αO-GeXIVA13, αO-GeXIVA14, versus α9α10 nAChR. In the diagrams B, C, D, abscissa refers to log value (Log [Toxin Concentration]M) of molar concentration (M) of αO-GeXIVA isomer; ordinate refers to does-response percentage (% Response), which is a ratio percentage of acetylcholine receptor current to control current under action of toxin of corresponding concentration, each dose-response percentage is a mean value (mean) of data of 6-12 Xenopus oocytes, and the curve shows standard error (SEM) at the same time.

(4) FIG. 3: shows concentration dose-response curves (A-E) of αO-GeXIVA12 to other nAChRs subtypes. In the figure, abscissa is log value (Log [Toxin Concentration]M) of molar concentration (M) of the used αO-GeXIVA12; ordinate is dose-response percentage (% Response), which is a ratio percentage of acetylcholine receptor current to control current under action of toxin of corresponding concentration, each dose-response percentage is a mean value (mean) of data of 6-12 Xenopus oocytes, and the curve shows standard error (SEM) at the same time. The figure indicates corresponding nAChRs subtype and half-blocking dose (IC.sub.50) for the subtype. The αO-GeXIVA12 shows diverse activity in blocking α*β4 and α*β2 nAChRs, has activity of blocking α*β2 nAChRs considerably higher than that of α*β4 nAChRs; and has similar activity in blocking mice muscle type nAChR (Mα1β1δε) and α7 nAChR subtypes.

(5) FIG. 4: shows concentration dose-response curves of 3 isomers, αO-GeXIVA12, GeXIVA13 and GeXIVA14, to α9α10 nAChR when conventional ND96 perfusate is replaced with barium ion DN96 perfusate (Ba.sup.++-ND96), in which abscissa refers to log value (Log [Toxin Concentration]M) of molar concentration (M) of the used 3 αO-GeXIVA isomers; ordinate is dose-response percentage (% Response), which is a ratio percentage of acetylcholine receptor current to control current under action of toxin of corresponding concentration, each dose-response percentage is a mean value (mean) of data of 9 Xenopus oocytes, and the curve shows standard error (SEM) at the same time. The figure also indicates half-blocking dose (IC.sub.50) of corresponding isomer to α9α10 nAChR subtype.

(6) FIG. 5: shows concentration dose-response curves of αO-GeXIVA12 to various NMDA receptor subtypes, in which abscissa refers to log value of molar concentration (M) of the used αO-GeXIVA12; ordinate is dose-response percentage (% Response), which is a ratio percentage of NMDA receptor current to control current under action of toxin of corresponding concentration, each dose-response percentage is a mean value (mean) of data of 3-5 Xenopus oocytes, and the curve shows standard error (SEM) of 95% confidence interval at the same time. The figure also indicates half-blocking dose (IC.sub.50) of corresponding NMDARs subtype to said subtype.

(7) FIG. 6: shows concentration dose-response curves (A-J) of 3 isomers, αO-GeXIVA12, αO-GeXIVA13 and αO-GeXIVA14, to various other subtypes of nAChR receptor, in which the signs have the same meanings of FIG. 3

(8) FIG. 7: shows concentration dose-response curves (A-F) of 3 isomers, αO-GeXIVA12, αO-GeXIVA13 and αO-GeXIVA14, to human nAChR receptor associated subtypes, in which the signs have the same meanings of FIG. 3.

(9) FIG. 8: shows concentration dose-response curves (A-K) of 3 isomers, αO-GeXIVA12, αO-GeXIVA13 and αO-GeXIVA14, to various mutant types of rat α9α10 nAChR receptor, in which the signs have the same meanings of FIG. 3.

(10) FIG. 9A-9C show that after αO-GeXIVA12 blocks α9α10 nAChR, the blocking effect of α-CTx RgIA [S4T; R9Cit; Y10Iodo, R11Q] on α9α10 nAChR can not be interrupted, which confirms that they bind to α9α10 nAChR at different sites. A. The arrow points at adminstration of 1 μM αO-GeXIVA12 at 5 min; B. The arrow points at administration of 20 nM α-CTx RgIA [S4T; R9Cit; Y10Iodo, R11Q] at 5 min; C. The arrow points at administration of 1 μM αO-GeXIVA12 1 min+[1 μM αO-GeXIVA12α20 nM α-CTx RgIA [S4T; R9Cit; Y10Iodo, R11Q] at 5 min].

(11) FIG. 10: shows the inhibition effects of a recombinant αO-superfamily conotoxin wild type GeXIVAWT (rCTx-K41) on Sf9 cells, in which abscissa is concentration of recombinant GeXIVAWT with unit of μg/ml, Control is a negative control without adding recombinant GeXIVAWT; ordinate is optical absorbance value measured with an enzyme-labeled immunoassay instrument (BIO-RAD MODEL 550) under 490 nm wavelength.

SPECIFIC MODELS FOR CARRYING OUT THE INVENTION

(12) The embodiments of the present invention are illustrated in conjunction with examples as follows. Those skilled in the art would understand the following examples are merely used for illustrating the present invention, rather than limiting the scope of the present invention. The specific technologies or conditions that are not given in the specification are carried out according to the technologies or conditions described in the documents in the art (e.g., Molecular Cloning: A Laboratory Manual, Edition 3, J. Sambrook, etc., translated by HUANG Peitang, etc., Science Press), corresponding reference documents or specifications. All reagents or instruments which manufacturers are not given are commercially available conventional products.

Example 1

Cloning and Sequence Analysis of New αO-Superfamily Conotoxin Gene Wild Type (GeXIVAWT)

(13) Conus generalis (C. generalis) living body was collected from coastal area of Hainan Island and Xisha Islands. Small amount column centrifugal tissue/cell total RNA extraction kit (Shanghai Huashun Bioengineering Co., Ltd.) was used to extract total RNA according its operation manual, then cDNA synthesis was performed. Specific steps were carried out according to documents (e.g., QUAN Yaru, LUO Sulan, LIN Qiujin, ZHANGSUN Dongting, ZHANG Ben, Studying on extraction of conotoxin RNA and synthesis of cDNA thereof, Chinese Journal of Marine Drugs, 2005, 24(2): 1-5).

(14) The above synthesized cDNA was used as template, a primer was designed according to untranslated region sequence of O1-gene superfamily precursor gene, and RT-PCR amplification was carried out to obtain specific PCR amplification product. The used primers were:

(15) TABLE-US-00001 Primer 1: (SEQ ID NO: 13) 5′-CATCGTCAAGATGAAACTGACGTG-3′; Primer 2: (SEQ ID NO: 14) 5′-CACAGGTATGGATGACTCAGG-3′.

(16) RT-PCR cycle program was: pre-denaturizing at 94° C. for 3 minutes, denaturizing at 94° C. for 30 seconds, annealing at 56° C. for 30 seconds, extending at 72° C. for 30 seconds, repeating for 30 cycles, then extending at 72° C. for 2 minutes.

(17) The above specific PCR product was recovered, linked to T-easy vector (Promega) and used to transform E. coli XL1 strain (other commercial competent E. coli cells could also be used), recombinants were picked out using blue and white colonies and ampicillin resistance, recombinant plasmids were extracted and purified and used for sequencing, different clones (e.g., 3-5 different clones) of same one PCR product were used for sequencing and analysis.

(18) Through sequence analysis and comparison, the cDNA gene of the O1-superfamily conotoxin new member, i.e., wild type propeptide GeXIVAWT, was obtained. The GeXIVAWT propeptide gene was analyzed with DNAStar software to obtain its open reading frame (ORF) sequence, i.e., SEQ ID NO: 1 and SEQ ID NO: 2, as follows:

(19) Open reading frame (ORF) encoding GeXIVAWT proprotein (having allele mutation, the framed 19.sup.th T and G referred to single base mutation sites thereof; the underlined parts referred to DNA sequence encoding mature peptide):

(20) TABLE-US-00002 (SEQ ID NO: 1) (SEQ ID NO: 2)

(21) The new member of O1-superfamily conotoxin encoded with the above sequence, i.e., wild type propeptide GeXIVAWT (also called as αO-conotoxin GeXIVAWT precursor or αO-GeXIVAWT precursor or GeXIVAWT precursor hereinafter), had the following amino acid sequence (the framed 7.sup.th L and V referred to amino acids of mutation sites, the underlines parts referred to amino acid sequence of signal peptide, arrow ↓ referred to post-translational modification processing site before and after mature peptide, italic referred to N-terminal propeptide region):

(22) TABLE-US-00003 (SEQ ID NO: 7) (SEQ ID NO: 8)

(23) The generated signal peptide and mature peptide encoded by precursor peptide cDNA gene of wild type GeXIVAWT were analyzed and predicted with online ProP 1.0 Server (Duckert, P.; Brunak, S.; Blom, N., Prediction of proprotein convertase cleavage sites. Protein engineering, design & selection: PEDS 2004, 17 (1), 107-12.).

(24) The nucleotide sequence encoding GeXIVAWT mature peptide was as follows (the framed parts referred codons encoding cysteines):

(25) TABLE-US-00004 (SEQ ID NO: 3)

(26) The amino acid sequence of GeXIVAWT wild type mature peptide (hereinafter also cited as αO-conotoxin GeXIVAWT or αO-GeXIVAWT or GeXIVAWT) was shown as follows (the framed parts referred to cysteines):

(27) TABLE-US-00005 (SEQ ID NO: 9)

(28) The wild type GeXIVAWT precursor peptide contained 3 regions: signal peptide, propeptide and mature peptide, the 7.sup.th site amino acid residue in the signal peptide was leucine or valine (L or V), corresponding codons were TTG or GTG. The wild type mature peptide region (SEQ ID NO: 9) contained 5 cysteines (Cys), which was different from all known conotoxins, and the comparison with the related gene superfamily members was shown in Table 1.

(29) TABLE-US-00006 TABLE 1 Comparison with conotoxin precursor protein sequences relating to αO-gene superfamily Super Name of Cysteine family peptide mode Precursor peptide sequence O.sub.1 αO-GeXIVA C-C-C-C-C {MKLTCV(L/V)IITVLFLTACQLTTA}VTYSRGEHKHRALMSTGTNYRLPK ↓ wild type TCRSSGRYCRSPYDC RRRYCRR ITDACV (SEQ ID NO: 15) αO-GeXIVA C-C-C-C {MKLTCV(L/V)IITVLFLTACQLTTA}VTYSRGEHKHRALMSTGTNYRLPK ↓ TCRSSGRYCRSPYDR RRRYCRR ITDACV (SEQ ID NO: 16) MVIIA C-C-CC-C-C {MKLTCVVIVAVLLLTACQLITA}DDSRGTQKHRALRSTTKLSTSTR↓[CKG KGAKCSRLMYDCCTGSCRSGKC]↓G (SEQ ID NO: 17) O.sub.2 TxVIIA C-C-CC-C-C {MEKLTILLLVAAVLMSTQALI}QSDGEKRQQAKINFLS.RKS↓TAESWWE GECKGWSVYCSWDWECCSGECTRYYCELW (SEQ ID NO: 18) O.sub.3 CaFr179 C-C-CC-C-C {MSGLGIMVLTLLLLVFMEA}SHQDAGEKQATQRDAINVRRRRSLARR↓[T VTEECEEDCEDEEKHCCNTNNGPSCARLCF]↓G (SEQ ID NO: 19) J PIXIVA C-C-C-C {MPSVRSVTCCCLLWMMFSVQLVTP}GSPGTAQLSGHRTAR↓]FPRPRIC NLACRAGIGHKYPFCHCR]↓GKRDAVSSSMAV (SEQ ID NO: 20) L LtXIVA C-C-C-C {MKLSVMFIVFLMLTMPMTCA}GISRSATNGGEADVRAHDKAANLMALLQ ER↓[MCPPLCKPSCTNC]↓G (SEQ ID NO: 21) A α-BuIA CC-C-C {MFTVFLLVVLTTTVVS}FPSDRASDGRNAAANDKASDVVTLVLK↓[GCCS TPPCAVLYC]↓GRRR (SEQ ID NO: 22) αA-PIVA CC-C-C-C-C {MFTVFLLVVLATTVV}SFTSDRASDDRNTNDKASRLLSHVVR↓[GCCGS YPNAACHPCSCKDRPSYCGQ]↓GR (SEQ ID NO: 23) C αc-PrXA C-C {MQTAYWVMVMMMVMWITAPLSEG}GKPKLIIRGLVPNDLTPQRILRSLI SGR↓[TYGIYDAKPPFSCAGLRGGCVLPPNLRPKFKE]↓GR (SEQ ID NO: 24) D αD-VxXXA C-CC-C-CC- {MPKLEMMLLVLLIFPLSYFIAAGG}QVVQVDRRGDGLAGYLQRGDR↓[D C-C-C-C VQDCQVSTPGSKWGRCCLNRVCGPMCCPASHCYCVYHRGRGHGCSC] (SEQ ID NO: 25) S αS-RVIIIA C-C-C-C-C- {MMSKMGAMFVLLLLFTLAS}SQQEGDVQARKTHPKREFQRILLRSGR↓ C-C-C-C-C [KCNFDKCKGTGVYNCGESCSCEGLHSCRCTYNIGSMKSGCACICTYY] (SEQ ID NO: 26)

(30) In the above table, characters between braces represent signal peptide amino acids, italic characters represent N-terminal propeptide region amino acids, characters within square brackets represent mature peptide amino acids, arrows “↓” represent post-translational modification processing sites before and after a mature peptide; and characters within a parenthesis represent amino acids of a mutation site.

Example 2

Preparation and Sequence Analysis of New αO-Superfamily Conotoxin Gene Mutant Type (GeXIVA)

(31) The 181.sup.st to 183.sup.rd bases TGC of the wild type GeXIVAWT precursor peptide gene encoded cysteine (Cys), they were subjected to point mutation (could also be obtained by direct artificial chemical synthesis of SEQ ID NO: 4), that was, single base mutated and became a codon CGC (GC.fwdarw.GC) for coding arginine Arg (R), i.e., the 181.sup.st site T was changed into C (T181C), this point mutant was named as GeXIVA precursor peptide gene, and its sequence was as follows:

(32) The open reading frame (ORF) (having allele mutation, the framed 19.sup.th T and G referred to its single base mutation site; the double underlined part referred to the 181.sup.st mutation site, and the underlined part referred to DNA sequence encoding mature peptide):

(33) TABLE-US-00007 (SEQ ID NO: 4) TCAACTCACTACAGCTGTGACTTACTCCAGAGGTGAGCATAAGCATCGTG CTCTGATGTCAACTGGCACAAACTACAGGTTGCCCAAGACGTGCCGTAGT TCCGGTCGTTATTGTCGCTCACCTTATGATCGCCGCAGAAGATATTGCAG ACGCATTACGGATGCGTGCGTATAG; or (SEQ ID NO: 5) TCAACTCACTACAGCTGTGACTTACTCCAGAGGTGAGCATAAGCATCGTG CTCTGATGTCAACTGGCACAAACTACAGGTTGCCCAAGACGTGCCGTAGT TCCGGTCGTTATTGTCGCTCACCTTATGATCGCCGCAGAAGATATTGCAG ACGCATTACGGATGCGTGCGTATAG.

(34) The above sequence coded new member of O1-superfamily conotoxin, i.e., mutant precursor peptide GeXIVA (hereinafter cited as αO-conotoxin GeXIVA precursor or αO-GeXIVA precursor or GeXIVA precursor) amino acid sequence (the framed 7.sup.th L and V referred to mutation site amino acid, the double underlined parts referred to the 61.sup.st site artificial mutation site, the underlined parts referred to the amino acid sequence of signal peptide, the arrow ↓ referred to post-translational modification processing site before and after mature peptide, italic referred to N-terminal propeptide region);

(35) TABLE-US-00008 (SEQ ID NO: 10) (SEQ ID NO: 11) 0

(36) The generated signal peptide and mature peptide encoded by precursor peptide cDNA gene of mutant GeXIVA were analyzed and predicted with online ProP 1.0 Server (Duckert, P.; Brunak, S.; Blom, N., Prediction of proprotein convertase cleavage sites. Protein engineering, design & selection: PEDS 2004, 17 (1), 107-12.).

(37) The nucleotide sequence encoding GeXIVA mature peptide was as follows (the framed parts referred codons encoding cysteines; the double underlined letter C referred to single base mutation site corresponding to point mutation amino acid):

(38) TABLE-US-00009 (SEQ ID NO: 6)

(39) The amino acid sequence of GeXIVA mutant mature peptide (hereinafter also cited as αO-conotoxin GeXIVA or αO-GeXIVA or GeXIVA) was shown as follows (the framed parts referred to cysteines; the double underlined letter R referred to point mutation amino acid):

(40) TABLE-US-00010 (SEQ ID NO: 12)

(41) The mutant GeXIVAWT precursor peptide contained 3 regions: signal peptide, propeptide and mature peptide, the 7.sup.th site amino acid residue in the signal peptide was leucine or valine (L or V), corresponding codons were TTG or GTG. The mutant mature peptide region (SEQ ID NO: 12) contained 4 cysteines (Cys), which was different from all known conotoxins, and the comparison with the related gene superfamily members was shown in the above Table 1.

(42) The following studying shows that GeXIVA was a blocking agent for nAChRs and NMDARs, and had strongest blocking activity to α9α10 nAChR, so it was formally named as αO-conotoxin GeXIVA, aliased as αO-GeXIVA or GeXIVA.

Example 3

Artificial Synthesis of αO-Conotoxin GeXIVA

(43) According to the amino acid sequence (SEQ ID NO: 12, C-terminal was not amidated) of αO-conotoxin GeXIVA mature peptide, the 3 possible isomer linear peptides GeXIVA12, GeXIVA13, GeXIVA14 (FIG. 1) of GeXIVA were artificially synthesized by Fmoc method. The specific method is as follows.

(44) The resin peptides of the 3 isomers were artificially synthesized by Fmoc chemical method, in which except cysteines, residual amino acids were protected with standard side chain protecting groups. As for GeXIVA12, the —SH groups of its 3.sup.rd and 4.sup.th cysteines (Cys) were protected with Trt (S-trityl), and the —SH groups of its 1.sup.st and 2.sup.nd cysteines (Cys) were protected with Acm (S-acetamidomethyl) in pairs; as for GeXIVA13, the —SH groups of its 2.sup.nd and 4.sup.th cysteines (Cys) were protected with Trt (S-trityl), and the —SH groups of its 1.sup.st and 3.sup.rd cysteines (Cys) were protected with Acm (S-acetamidomethyl) in pairs; as for GeXIVA14, the —SH groups of its 2.sup.nd and 3.sup.rd cysteines (Cys) were protected with Trt (S-trityl), and the —SH groups of its 1.sup.st and 4.sup.th cysteines (Cys) were protected with Acm (S-acetamidomethyl) in pairs. The synthesis steps comprise: using Fmoc and FastMoc methods of solid phase synthesis method, synthesizing 3 isomer linear peptides by ABI Prism 433a polypeptide synthesizer. The side chain protecting groups of Fmoc amino acids were: Pmc (Arg), Trt (Cys), But (Thr, Ser, Tyr), OBut (Asp), Boc (Lys). Fmoc HOBT DCC method was used, and its steps were carried out according to synthesis manual of instrument. In order to complete synthesis, piperidine deprotecting time and coupling time were properly extended, respectively, double coupling was used for amino acids difficult to link, and thus the resin peptides were obtained. The linear peptide was cut from resin using reagent K (trifluoroacetic acid/water/ethanedithiol/phenol/thioanisole; 90:5:2.5:7.5:5,v/v/v/v/v), and subjected to ice diethyl ether precipitation and washing to recover a crude product of the linear peptide, preparative reversed phase HPLC C18 column (Vydac) was used for purification, and elution linear gradient was 10-50% B60 within 0-40 min. Solution B was 60% ACN (acetonitrile), 40% H20, 0.92% TFA (trifluoroacetic acid); solution A was 1% TFA aqueous solution.

(45) The purified linear peptide was subjected to purity detection with HPLC C18 column (Vydac), showing its purity was 96% or more, then it could be used for oxidation folding. The linear peptides of 3 isomers, GeXIVA12, GeXIVA13 and GeXIVA14, were subjected to two-step oxidation folding reaction according to documents (Dowell, C.; Olivera, B. M.; Garrett, J. E.; Staheli, S. T.; Watkins, M.; Kuryatov, A.; Yoshikami, D.; Lindstrom, J. M.; McIntosh, J. M., Alpha-conotoxin PIA is selective for alpha6 subunit-containing nicotinic acetylcholine receptors. The Journal of neuroscience 2003, 23 (24), 8445-52.), and the process thereof was briefly described as follows:

(46) Firstly, the first pair of disulfide bond between two cysteines with Trt protecting groups was formed by potassium ferricyanide method (20 mM potassium ferricyanide, 0.1 M Tris, pH 7.5, 30 min). After monocycle peptide was purified with reversed phase HPLC C18 column (Vydac), iodine oxidation was carried out (10 mM iodine in H.sub.2O:trifluoroacetic acid:acetonitrile (78:2:20 by volume, 10 min), to remove Acm of another 2 cysteines, and form the second pair of disulfide bond between the 2 cysteines at the same time. Dicyclic peptide was purified with reversed phase HPLC C18 column (Vydac) to obtain αO-conotoxin in which disulfide bonds were directionally formed between corresponding cysteines in sequence of N-terminal to C-terminal, and confirmed with mass spectrum (MS). The theoretical molecular weight (monoisotopic mass) of the 3 isomers after oxidation folding was 3451.96 Da, the measured molecular weight of GeXIVA12 was 3451.83 Da; the measured molecular weight of GeXIVA13 was 3451.72 Da; and the measured molecular weight of GeXIVA14 was 3452.05 Da. Colorimetric assay was used to detect polypeptide concentration under wavelength of 280 nm, and polypeptide concentration and mass were calculated according to Beer-Lambert (equation). These quantified isomers were continuously used for subsequent activity assay (e.g., Example 5-10).

Example 4

Expression of Rat, Mice and Human nAChRs Subtypes in Xenopus Oocytes

(47) The method of document (Azam L, Yoshikami D, McIntosh J M. Amino acid residues that confer high selectivity of the alpha6 nicotinic acetylcholine receptor subunit to alpha-conotoxin MII[S4A,E11A,L15A]. J Biol Chem. 2008; 283(17):11625-32.), the specification of in vitro transcription kit (mMessage mMachine in vitro transcription kit (Ambion, Austin, Tex.)) were referred to prepare various rat nervous type nAChRs subtypes (α3β2, α6/α3β2β3, α6/α3β4, α9α10, α4β2, α4β4, α3β4, α2β2, α2β4, α7), human nervous type nAChRs subtypes (α9α10, α6/α3β2β3, α7), various mutants of rat α9α10 nAChR, and cRNA of mice and human muscle type nAChRs (α1β1δε), their concentrations were measured and calculated by OD values under UV 260 nm. The oocytes (frogspawns) of Xenopus (Xenopus laveis) were collected and dissected, cRNA was injected into frogspawns, the injection dose for each subtype was 5 ng cRNA. For muscle nAChR, each subtype was injected with 0.5-2.5 ng DNA. The frogspawns were cultured in ND-96. The collected frogspawns were injected with cRNA within 1-2 days, and used for nAChRs voltage clamp recording within 1-4 days after the injection. Ba.sup.2+-contained ND-96 buffer solution was obtained by replacing CaCl.sub.2 with BaCl.sub.2 at equivalent molar concentration. The prepared samples were used for example in following Examples 5-10.

Example 5

Experiment of Blocking Various Rat nAChRs with 3 Isomers of αO-Conotoxin GeXIVA

(48) One of frogspawns injected with cRNA was placed in 30 uL of Sylgard record tank (diameter 4 mm×depth 2 mm), gravity perfused with ND96 perfusate (96.0 mM NaCl, 2.0 mM KCl, 1.8 mM CaCl.sub.2, 1.0 mM MgCl.sub.2, 5 mM HEPES, pH 7.1-7.5) containing 0.1 mg/ml BSA (bovine serum albumin), or ND96 (ND96A) containing 1 mM atropine, flow rate was 1 ml/min. All conotoxin solutions also contained 0.1 mg/ml BSA to reduce non-specific adsorption of toxin, a change-over valve (SmartValve, Cavro Scientific Instruments, Sunnyvale, Calif.) could be used for freely switching between perfusion of toxin and acetylcholine (ACh), and a series of three-way solenoid valves (solenoid valves, model 161TO31, Neptune Research, Northboro, Mass.) were used for freely switching between perfusion of ND96 and ACh. Ach gating current was set at “slow” clamp with double-electrode voltage clamp amplifier (model OC-725B, Warner Instrument Corp., Hamden, Conn.), and on-line recording of clamp gain was performed at the maximum value (×2000) position. Glass electrodes were drawn from glass capillaries (fiber-filled borosilicate capillaries, WPI Inc., Sarasota, Fla.) with 1 mm external diameter×0.75 mm internal diameter, and filled with 3 M KCl as voltage and current electrodes. Membrane voltage was clamped at −70 mV. The control of whole system and data recording were carried out with a computer. ACh pulse was automatically perfusing ACh for 1 s per interval of 5 min. ACh had concentration of 10 μM for oocytes of muscle type nAChRs and nervous type α9α10 nAChRs; 200 μM for α7 of nervous type nAChRs, and 100 μM for other subtypes. At least 4 oocytes were used for recording situations of current response and current track of a subtype under different toxin concentrations.

(49) The measured current data were subjected to statistic analysis with GraphPad Prism software (San Diego, Calif.), dose-response curves were plotted, half-blocking concentration (IC.sub.50) of conotoxin and many other parameters relating to toxin-blocking nAChRs were calculated.

(50) The results shown that 33 nM αO-GeXIVA12 (prepared in Example 3) completely blocked the current generated by Ach-gated α9α10 nAChR open, and had features of fast elution and reversible blocking (FIG. 2A). All of 3 isomers had strong blocking activity to α9α10 nAChR. Among the 3 isomers, αO-GeXIVA12 had the strongest activity, αO-GeXIVA14 took the second place, and αO-GeXIVA13 had the weakest activity (FIG. 2 B, C, D). Their half-blocking doses (IC.sub.50) and error ranges separately were: GeXIVA12, 4.6 nM (3.18-6.65 nM); GeXIVA13, 22.7 nM (11.8-43.5 nM); GeXIVA14, 7 nM (3.6-13.4 nM). The dose-response curves of the 3 isomers separately had slopes (Hillslope) and error ranges as follows: GeXIVA12, 0.56(0.44-0.69); GeXIVA13, 0.78(0.29-1.26), GeXIVA14, 0.79(0.23-1.36). The αO-GeXIVA12 had different blocking activity on various nAChRs subtypes, and its half-blocking doses IC.sub.50 and slopes of dose-response curves were shown in Table 2.

(51) TABLE-US-00011 TABLE 2 Half-blocking doses IC.sub.50 and slopes of dose-response curves of αO-GeXIVA12 to various nAChRs subtypes nAChRs Half-blocking dose Ratio to half-blocking Slope of subtype IC.sub.50 (nM).sup.a dose of α9α10 subtype.sup.b dose-response curve.sup.3 α9α10  4.61 (3.18-6.65) 1 0.56 (0.44-0.69) α7 415 (264-655) 90.0 1.12 (0.68-1.56) Mouse α1β1δε 394 (311-498) 85.5 1.71 (0.98-2.43) α6/α3β2β3 258 (200-331) 56.0 0.63 (0.53-0.72) α6/α3β4  806 (453-1140) 175.2 1.18 (0.57-1.79) α2β2 338 (178-640) 73.3 1.133 (0.33-1.94)  α2β4  2090 (1430-3070) 454.3 1.12 (0.73-1.51) α3β2 412 (223-761) 89.6 1.033 (0.37-1.70)  α3β4   5400 (3390- to 8580) 1171 1.12 (0.47-1.77) α4β2  979 (672-1425) 212.4 0.083 (0.51-0.86)  α4β4  2390 (1560-3670) 662.9 0.21 (0.67-1.65) Notation: .sup.avalue in bracket had confidence interval of 95%; .sup.breferred to nAChR subtype IC.sub.50 /α9α10 IC.sub.50;

(52) The difference of blocking activity of αO-GeXIVA12 to α*β4 and α*β2 nAChRs was relatively great, the blocking activity to α*β2 nAChRs was far greater than that to α*β4 nAChRs (FIG. 3, A-E); the blocking activity to mice muscle type nAChR (Mα1β1βε) and to α7 nAChR subtype were relatively close. It was shown that αO-GeXIVA12 more preferably blocked β2-containing nAChRs, including α6/α3β2β3, α4β2, α3 β2 and α2β2. The blocking activity of αO-GeXIVA12 to α9α10 nAChR was at least 56-663 times higher than that to other subtypes. Under a low concentration of less than 200 nM, αO-GeXIVA12 was a specific blocking agent for α9α10 nAChR, but had very weak or almost not blocking activity to other nAChRs subtypes. The comparison of action target biological activity of αO-GeXIVA12 and other superfamily conotoxins was shown in Table 3.

(53) TABLE-US-00012 TABLE 3 Comparison of properties between αO-GeXIVA and other conotoxins Name of toxin Molecular peptide Kind of source Sequence Target weight αO-GeXIVA C. generalis TCRSSGRYCRSPYDRRRRYC α9α10 nAChR >> α*β2 3452 RRITDACV{circumflex over ( )} nAChRs >> α*β4 nAChRs nAChRs > NMDAR ω-MVIIA C. magus CKGKGAKCSRLMYDCCTGSC Cav2.2 > Cav2.1 2637 RSGKC# N > P/Q pI14a (κJ-PIXIVA) C. planorbis FPRPRICNLACRAGIGHKYPF Kv1.6 > 1.1 > 2909 pI14a (αJ-PIXIVA) CHCR# 1.2~1.3~1.4~1.5~2.1~3.4 Muscle > α3β2 nAChRs It14a (αL-LtXIVA) C. litteratus MCPPLCKPSCTNC# Neuronal nAChR 1391 vi114a (KL-VilXIVA) C. Villepinii GGLGRCIYNCMNSGGGLSFIQ N.D. 2871 CKTMCY{circumflex over ( )} ψ-PIIIE C. HOOCCLYGKCRRYOGCSSAS Muscle nAChR 2715 purpurascens CCQR# α-LtIA C. litteratus GCCARAACAGIHQELC# α3β2 > a6/α3β2β3 1600 αA-EIVA C. ermineus GCCGPYONAACHOCGCKVG Muscle nAChR 3094 ROOYCDROSGG# αC-PrXA C. parius TYGIYDAKPOFSCAGLRGGCV Muscle nAChR 3539 LPONLROKFKE# αD-VxXXB C. vexillum DDγSγCIINTRDSPWGRCCRT α7 > α3β2 5735 RMCGSMCCPRNGCTCVYHW RGHGCSCPG (dimer) αS-RVIIIA C. radiatus KCNFDKCKGTGVYNCG(Gla)S Muscle nAChR 5167 CSC(Gla)GLHSCRCTYNIGSM Neuronal nAChR KSGCACICTYY

(54) It was known that α9α10 nAChR has very high permeability to calcium ion (Ca.sup.++). The calcium ion internal flow of nAChRs could activated the generation of chlorine ion (Cl.sup.−) external flow, at for xenopus oocytes, this kind of current was 90% or more of the observed α9α10nAChR open current. On the contrary, barium ion (Ba.sup.++) that was close to calcium ion did not activate chlorine ion current. Thus, we used barium ion ND96 perfusate (Ba.sup.++-ND96, 1.8 mM BaCl.sub.2 replaced CaCl.sub.2) to replace conventional ND96 perfusate, and found that the observed open current of α9α10nAChR was far lower than that of the conventional ND96 perfusate, which was consistent with the previous studying. Under condition of barium ion ND96 perfusate, GeXIVA12 showed the strongest blocking activity to α9α10nAChR, GeXIVA14 took the second place, and the activity of GeXIVA13 was the weakest (FIG. 4). As for the isomer αO-GeXIVA12 having I-II and III-IV disulfide bonds, its half-blocking dose (IC.sub.50) to α9α10 nAChR and error range thereof were 3.8 nM (3.1-4.8 nM), curve slope (Hillslope, nH) and error range thereof were 0.71 (0.58-0.84); as for the isomer αO-GeXIVA13 having I-III and II-IV disulfide bonds, its half-blocking dose (IC.sub.50) to α9α10 nAChR and error range thereof were 37 nM (25.0-55.7 nM), curve slope (Hillslope) and error range thereof were 0.54 (0.42-0.65); as for the isomer αO-GeXIVA14 having I-IV and II-III disulfide bonds, its half-blocking dose (IC.sub.50) to α9α10 nAChR and error range thereof were 5.8 nM (4.7-7.1 nM), curve slope (Hillslope) and error range thereof were 0.65 (0.56-0.73). Under condition of barium ion ND96 perfusate, the 3 isomers of αO-GeXIVA had results of activity similar to those under calcium ion-containing normal ND96 perfusate. In addition, under barium ion-ND96, the activities of αO-GeXIVA12 and αO-GeXIVA14 were stronger than those under calcium ion-containing normal ND96, while the activity of αO-GeXIVA13 under barium ion-ND96 was stronger than that under normal ND96. Thus, the 3 isomers of GeXIVA did block α9α10nAChR, instead of blocking chlorine ion current that was activated due to calcium ion.

Example 6

Experiment of 3 Isomers of αO-Conotoxin GeXIVA Blocking Rat NMDA Receptor

(55) The method of document (Twede, V. D. et al. Conantokin-Br from Conus brettinghami and selectivity determinants for the NR2D subunit of the NMDA receptor. Biochemistry, 2009, 48: 4063-4073) was referred to, and method similar to expression of nAChRs in xenopus oocytes was used to prepare cRNAs corresponding to 4 subtypes of rat NMDA receptor: NR1-2b/NR2A, NR1-2b/NR2B, NR1-2b/NR2C, NR1-2b/NR2D, which concentrations were measured and calculated with OD values under UV 260 nm. The oocytes (frogspawns) of Xenopus (Xenopus laveis) were collected and dissected, cRNA was injected into frogspawns, the injection dose for each subtype was 5 ng cRNA. The frogspawns were cultured in ND-96. The collected frogspawns were injected with cRNA within 1-2 days, and used for nAChRs voltage clamp recording within 1-4 days after the injection. Voltage clamp recording NMDA receptor current was carried out by a method similar to that of nAChRs, except that the used perfusate was magnesium-free ND96 perfusate (Mg.sup.2+-free ND96 buffer), which composition comprised 96.0 mM NaCl, 2.0 mM KCl, 1.8 mM CaCl2, 5 mMHEPES (pH 7.2-7.5). The reason was that Mg.sup.2+ could block NMDA receptor under −70 mV clam voltage. The NMDA receptor agonist solution was Mg.sup.2+-free ND96 that contained 200 μM glutamate and 20 μM glycine in final concentration. The αO-GeXIVA12 had the strongest blocking activity to NR2C NMDAR subtype, and can be rapidly eluted. As for αO-GeXIVA12, its half-blocking doses (IC.sub.50) and error range thereof to 4 kinds of NMDA receptor subtypes separately were: NR2C, 0.66 μM (0.38-1.1 μM); NR2B, 4.0 μM (2.2-7.3 μM); NR2A, 3.7 μM (2.8-5.0 μM); NR2D, 5.2 μM (1.7-15.7 μM); as for αO-GeXIVA12, its dose-response curve slopes (Hillslope) and error ranges thereof to 4 kinds of NMDA receptor subtypes separately were: NR2C, 0.13 (0.42-0.97); NR2B, 0.22(0.36-1.3); NR2A, 0.10 (0.61-1.07); NR2D, 0.15 (0.10-0.78) (FIG. 5). The half-blocking doses (IC.sub.50) and dose-response curve slopes of 3 isomers of αO-GeXIVA to subtypes of various NMDARs were shown in Table 4.

(56) TABLE-US-00013 TABLE 4 Half-blocking doses (IC.sub.50) and dose-response curve slopes of 3 isomers of αO-GeXIVA to subtypes of various NMDARs NMDARs Isomer subtype NR2A NR2B NR2C NR2D GeXIVA12 IC.sub.50 (nM).sup.a  3700 (2800-5000) 4000 (2200-7300)  655 (380-1100) 5200 (1700-15700) slope 0.84 (0.61-1.07) 0.85 (0.36-1.33)  0.69 (0.42-0.97) 0.44 (0.10-0.78)   GeXIVA13 IC.sub.50 (nM) >10000 >10000 (17000)     ≧10000 ≧10000 slope — — — — GeXIVA14 IC.sub.50 (nM)   7400 (3800-14000) 3400 (1500-7700)  >10000 ≧10000 slope 1.08 (0.15-2.01) 0.94 (0.28-1.60)  — — Notation: .sup.avalue in bracket had confidence interval of 95%.

(57) The activities of αO-GeXIVA12 to 4 kinds of NMDA receptor subtypes were in sequence from strong to weak as follows: NR2C>NR2A>NR2B>NR2D. The αO-GeXIVA14 had relatively weak blocking activity to 2 kinds of NMDA receptor subtypes NR2B and NR2A, while αO-GeXIVA13 had very weak or even no blocking activity to 4 kinds of NMDA receptor subtypes.

Example 7

Experiments of 3 Isomers of αO-Conotoxin GeXIVA Blocking Other Rat nAChRs and Mice Muscle Type nAChRs

(58) The same experimental methods of Examples 4 and 5 were used to study the effects of 3 isomers of αO-conotoxin GeXIVA on blocking other rat nAChRs and mice muscle type nAChRs. The concentration dose-response curves of 3 isomers, αO-GeXIVA12, αO-GeXIVA13 and αO-GeXIVA14, to other subtypes of nAChR receptors were shown in FIG. 6 (A-J). Generally, the blocking activates of the 3 isomers to α*β2 nAChRs were higher that those to α*β4 nAChRs (FIG. 6); the blocking activities of 3 isomers to mice muscle type nAChR (Mα1β1δε) were similar, and their half-blocking doses (IC.sub.50) separately were: αO-GeXIVA12, 394 nM; αO-GeXIVA13, 671 nM; αO-GeXIVA14, 473 nM. However, the blocking activities of the 3 isomers to α7 nAChR subtype were greatly diverse, the order of their IC.sub.50 activities was αO-GeXIVA12, 415 nM>αO-GeXIVA14, 1740 nM>αO-GeXIVA13, 4960 nM. The activity order of αO-GeXIVA12>GeXIVA14>GeXIVA13 also occurred in mice muscle type nAChR and nervous type α3β2 nAChR, but the diversity of blocking activities between the 3 isomers to Mα1β1δε and α3β2 nAChRs was very small.

(59) As for the blocking activities to α2β2, α2β4, α4β2, α4β4 nAChRs, the blocking activity of GeXIVA14 was the strongest, the IC.sub.50 to α2β2 was merely 122 nM, the IC.sub.50 to α4β2 was 200 nM. As for the blocking activities to α3β4, α6/α3β4 nAChR, the blocking activity of GeXIVA13 was the strongest, which IC.sub.50 was 483 nM; GeXIVA14 took the second place, which IC.sub.50 was 611 nM; while GeXIVA12 showed the weakest blocking activity, which IC.sub.50 was 806 nM. However, the blocking activities of the 3 isomers to all other nAChRs subtypes were far less than that to α9α10 nAChR. The differential blocking activities of the 3 isomers to different nAChRs subtypes provided a theoretical basis for designing a series of selective blocking agents using GeXIVA as template for different subtypes.

Example 8

Experiment of 3 Isomers of αO-Conotoxin GeXIVA Blocking Human nAChRs

(60) The same experimental methods of Examples 4 and 5 were used to study the effects of 3 isomers of αO-conotoxin GeXIVA on blocking human nAChRs. Among the 3 isomers, both αO-GeXIVA12 and GeXIVA14 showed very strong blocking activities to human α9α10 nAChR, αO-GeXIVA12 had the strongest activity, αO-GeXIVA14 took the second place, and αO-GeXIVA13 showed the weakest activity (FIG. 7, A-F). Their half-blocking doses (IC.sub.50) and error range thereof separately were: GeXIVA12, 20 nM (12.4-33.2 nM); GeXIVA13, 116 nM (65.4-204 nM); GeXIVA14, 47 nM (29.7-75.3 nM). The slopes of dose-response curves (Hillslope) of the 3 isomers and error ranges thereof separately were: GeXIVA12, 0.91(0.49-1.32); GeXIVA13, 0.73(0.45-1.01), GeXIVA14, 0.67(0.46-0.88). The blocking activities of the 3 isomers to human muscle type nAChR (Human α1β1δε) were similar, and their half-blocking doses (IC.sub.50) separately were: αO-GeXIVA12, 497 nM; αO-GeXIVA13, 485 nM; αO-GeXIVA14, 365 nM. However, the blocking activities of the 3 isomers to α7 nAChR subtype were very diverse, and their IC.sub.50 activity order was αO-GeXIVA12, 555 nM>αO-GeXIVA14, 865 nM>αO-GeXIVA13, 3300 nM. The blocking activity order to human α9α10 and α7 nAChRs, αO-GeXIVA12>GeXIVA14>GeXIVA13, was consistent to the blocking activity order to mice α9α10 and α7 nAChRs. However, the blocking activity order of the 3 isomers to human α6/α3β2β3 nAChR was αO-GeXIVA13≧GeXIVA14>GeXIVA12, and their half-blocking doses (IC.sub.50) separately were: αO-GeXIVA13, 141 nM; αO-GeXIVA14, 197 nM; αO-GeXIVA12, 505 nM. The differential blocking activities of the 3 isomers to different human nAChRs subtypes were advantageous to design a series of αO-GeXIVA analogues so as to obtain selective blocking agents for different subtypes.

Example 9

Experiment of 3 Isomers of αO-Conotoxin GeXIVA Blocking Various Mutants of Rat α9α10 nAChR Receptor

(61) The possible key amino acids of wild type α9α10 nAChR receptor at sites binding to conotoxin were subjected to point mutation, i.e., change of single amino acid, to prepare various mutants of α9α10 nAChR receptor. The mutation sites were shown in FIG. 8, for example, “rα9R71Gα10” referred to that in α9 subtype of rat (r) wild type α9α10 nAChR receptor, the 71.sup.st arginine R(Arg) was mutated into glycine G(Gly), and the representing methods of other mutants were in the same manner. The receptor mutants were prepared by PCR method, i.e., subjecting the codons corresponding to point mutation amino acids in gene of α9 or α10 subtype of wild type α9α10 nAChR receptor to mutation. As for the obtained mutants, the effects of 3 isomers of αO-conotoxin GeXIVA on blocking various α9α10 nAChRs mutants were studied according to the same experimental methods of Examples 4 and 5 (FIG. 8).

(62) The blocking activities of the 3 isomers, αO-GeXIVA12, αO-GeXIVA13 and αO-GeXIVA14, to 11 rat α9α10 nAChR receptor mutants were tested. Their concentration dose-response curves were shown in FIG. 8 (A-K). The general trend was that all of the 3 isomers had very strong blocking activity to various mutants of α9α10 nAChR, the blocking activities of αO-GeXIVA12 and αO-GeXIVA14 to various mutants were similar, and stronger than the activity of αO-GeXIVA13, which was in consistence with the blocking activity order of the wild type α9α10 nAChR. The blocking activity order of the 3 isomers was αO-GeXIVA12≧GeXIVA14>GeXIVA13. There were 5 mutants having relatively great change of activity, the half-blocking dose IC.sub.50 of αO-GeXIVA12 was 46-59 nM; the half-blocking dose IC.sub.50 of αO-GeXIVA14 was 34-96 nM; the half-blocking dose IC.sub.50 of αO-GeXIVA13 was 106-232 nM. The half-blocking doses IC.sub.50 of the 3 isomers to these 5 α9α10 nAChR mutants separately were: (1) rα9R71Gα10, αO-GeXIVA12, 59 nM; αO-GeXIVA13, 232 nM; αO-GeXIVA14, 61 nM; (2) α9S14Nα10, αO-GeXIVA12, 53 nM; αO-GeXIVA13, 108 nM; αO-GeXIVA14, 60 nM; (3) α9A24Kα10, αO-GeXIVA12, 52 nM; αO-GeXIVA13, 170 nM; αO-GeXIVA14, 96 nM; (4) α9E192Qα10, αO-GeXIVA12, 50 nM; αO-GeXIVA13, 143 nM; αO-GeXIVA14, 63 nM; (5) α9S136Nα10, αO-GeXIVA12, 46 nM; αO-GeXIVA13, 106 nM; αO-GeXIVA14, 34 nM.

(63) There were 6 mutants having very small change of activity, the half-blocking dose IC.sub.50 of αO-GeXIVA12 was 14-32 nM; the half-blocking dose IC.sub.50 of αO-GeXIVA14 was 24-55 nM; the half-blocking dose IC.sub.50 of αO-GeXIVA13 was 68-182 nM. As for 2 mutants, rα9S136Nα10 and rα9α10E56S, the blocking activity order of the 3 isomers was αO-GeXIVA14≧GeXIVA12>GeXIVA13. As for rα9S136Nα10 mutant, αO-GeXIVA14 (IC.sub.50, 34 nM) showed stronger blocking activity than GeXIVA12 (IC.sub.50, 46 nM). The half-blocking dose IC.sub.50 of the 3 isomers to these 6 α9α10 nAChR mutants separately were: (1) rα9S117Aα10, αO-GeXIVA12, 32 nM; αO-GeXIVA13, 182 nM; αO-GeXIVA14, 47 nM; (2) αα10D116L, αO-GeXIVA12, 28 nM; αO-GeXIVA13, 122 nM; αO-GeXIVA14, 45 nM; (3) α9α10E56S, αO-GeXIVA12, 25 nM; αO-GeXIVA13, 68 nM; αO-GeXIVA14, 24 nM; (4) α9T56Iα10, αO-GeXIVA12, 23 nM; αO-GeXIVA13, 76 nM; αO-GeXIVA14, 33 nM; (5) α9S6Nα10, αO-GeXIVA12, 20 nM; αO-GeXIVA13, 119 nM; αO-GeXIVA14, 55 nM; (6) α9T64Iα10, αO-GeXIVA12, 14 nM; αO-GeXIVA13, 79 nM; αO-GeXIVA14, 24 nM.

(64) The mutation sites of these α9α10 nAChR mutants were key amino acids of the receptor that were previously found to bind to α-conotoxin (Ellison M, Feng Z P, Park A J, Zhang X, Olivera B M, McIntosh J M, Norton R S. Alpha-RgIA, a novel conotoxin that blocks the alpha9alpha10 nAChR: structure and identification of key receptor-binding residues. J Mol Biol. 2008; 377(4):1216-27), the 3 isomers of αO-GeXIVA had not significant influence on activities of these mutants, i.e., their activity changes were about 10 or less times that of wild type α9α10 nAChR. This indicated that the binding sites or parts of αO-GeXIVA to α9α10 nAChR were totally different from those previously disclosed binding sites, i.e., they were new action sites.

Example 10

Novel Sites of αO-Conotoxin GeXIVA12 Specifically Blocking α9α10 nAChR

(65) The elution of αO-GeXIVA12 blocking α9α10 nAChR was very fast (FIG. 9A). α-CTx RgIA [S4T; R9Cit; Y10Iodo, R11Q] (or RgIAM for short) was a specific blocking agent for α9α10 nAChR, but its elution was recovered very slowly (confirmed with experiment), that was, when toxin RgIAM was eluted, α9α10 nAChR restored very slowly to ACh normal gating open state (FIG. 9B).

(66) According to the different elution rates of the two, the inventors of the present invention designed a competitive test (FIG. 9C). That was, after incubation with 1 μM αO-GeXIVA12 to block α9α10 nAChR for 1 min, 20 nM α-CTx RgIAM and 1 μM αO-GeXIVA12 were then used in the same cell tank of frogspawn to continue incubation for blocking α9α10 nAChR 5 min, Ach gating current record showed its elution rate was very slow, and this was the same situation of solely using 20 nM α-CTx RgIAM for elution. In the meantime, it was set to use ND96 to separately replace αO-GeXIVA12 and α-CTx RgIAM, as positive and negative controls. The results showed that αO-GeXIVA12 could not inhibit the blocking activity of α-CTx RgIA M to α9α10 nAChR, which confirmed that the binding sites of the two to α9α10 nAChR were totally different, αO-conotoxin GeXIVA12 bound to novel sites of α9α10 nAChR, which were different from the previously disclosed binding sites of α-conotoxin, and not overlapped.

(67) The studying showed that α9α10 nAChR were new target for treatment of neuralgia, chemical therapy of cancers, breast cancer, lung cancer, wound healing (McIntosh, J. M.; Absalom, N.; Chebib, M.; Elgoyhen, A. B.; Vincler, M., Alpha9 nicotinic acetylcholine receptors and the treatment of pain. Biochemical pharmacology 2009, 78 (7), 693-702. Satkunanathan, N.; Livett, B.; Gayler, K.; Sandall, D.; Down, J.; Khalil, Z., Alpha-conotoxin Vc1.1 alleviates neuropathic pain and accelerates functional recovery of injured neurones. Brain research 2005, 1059 (2), 149-58. Holtman, J. R.; Dwoskin, L. P.; Dowell, C.; Wala, E. P.; Zhang, Z.; Crooks, P. A.; McIntosh, J. M., The novel small molecule alpha9alpha10 nicotinic acetylcholine receptor antagonist ZZ-204G is analgesic. European journal of pharmacology 2011, 670 (2-3), 500-8. Zheng, G.; Zhang, Z.; Dowell, C.; Wala, E.; Dwoskin, L. P.; Holtman, J. R.; McIntosh, J. M.; Crooks, P. A., Discovery of non-peptide, small molecule antagonists of alpha9alpha10 nicotinic acetylcholine receptors as novel analgesics for the treatment of neuropathic and tonic inflammatory pain. Bioorganic & medicinal chemistry letters 2011, 21 (8), 2476-9. Chernyaysky, A. I.; Arredondo, J.; Vetter, D. E.; Grando, S. A., Central role of alpha9 acetylcholine receptor in coordinating keratinocyte adhesion and motility at the initiation of epithelialization. Experimental cell research 2007, 313 (16), 3542-55; Chikova, A.; Grando, S. A., Naturally occurring variants of human Alpha9 nicotinic receptor differentially affect bronchial cell proliferation and transformation. PloS one 2011, 6 (11), e27978.). Hence, the new αO-superfamily conotoxin GeXIVA of the present invention is very promising in mechanism studying, diagnosis, and treatment of the above diseases.

Example 11

Experiment of Recombinant αO-Conotoxin GeXIVAWT Inhibiting Sf9 Cell Growth

(68) The gene of wild type toxin (αO-GeXIVAWT) was inserted into between restriction enzyme cutting sites Nco I and Xho I of E. coli expression vector pET22b(+), to construct a fusion protein expression vector which N-terminal fused with pelB leader and C-terminal fused with His-tag purification label. The αO-GeXIVAWT recombinant protein was separated and purified. The effects of the recombinant conotoxin αO-GeXIVAWT on the growth state of Sf9 cells (Spodoptera frugiperda 9 (Sf9) cells, purchased from Invitrogen Company of USA) was studied by MTT method (FIG. 10). The method was carried out according to that MTT could penetrated cell membrane and entered into cell, succinate dehydrogenase in a living cell mitochondria could reduce exogenous MTT into water-insoluble needle-like blue-purple formazane crystal which precipitated in the cell, while dead cell had not such function. Dimethylsulfoxide (DMSO) could dissolve the blue-purple crystal in cell, and the intensity of color of the resultant solution was in direct proportion to the contained formazane content. Its optical density value (OD value) was measured under wavelength of 570 nm with ELIASA, and could indirectly reflect number of cells. The results showed that αO-GeXIVAWT could significantly inhibit growth of Sf9 cells, had dose effect, and could kill Sf9 cells under high concentration (>10 μg/ml). Sf-9 insect cell line was from ovary cell line Sf-21 of agricultural insect Spodoptera Frugiperda, this insect cell was very prone to infection with alfalfa California nuclear polyhedrosis virus (AcMNPV baculovirus) as biopesticide, and could be used as expression vector for all baculovirus. Hence, the wild type recombinant conotoxin αO-GeXIVAWT was promising in pest control (Bruce C, Fitches E C, Chougule N, Bell H A, Gatehouse J A (2011) Recombinant conotoxin, TxVIA, produced in yeast has insecticidal activity. Toxicon 58:93-100.).

(69) Although the embodiments of the present invention are described in details, those skilled in the art would understand that these details could be modified and changed according to the disclosed teachings, and all these changes fall into the protection scope of the present invention. The whole scope of the present invention is given by the appended claims and any equivalents thereof.