Conopeptides (Conotoxin) derived from tropical marine gastropod, cone snail ( Conus ) venoms have been an useful tool agent in neuro investigation and new drug source. Recently, new method of Conotoxin genes cloning from Conus genomic DNA has been established. So to get Conus genomic DNAs rapidly is the basis for diverse Conotoxin gene isolation, gene bank construction and the medical source exploitation. In this report, different DNA extractions from different tissue and organs of six cone snail species were performed in order to optimize the DNA isolation methods. The optimized brief phenol SDS method was established which fit for cone snails genomic DNA extraction.

Two methods of different RNA extractions from Conus venom tube and toxin gland of seven cone snail species were performed. ThermoScript~ TM RNase H~ - Transcriptase and AMV Transcriptase were used to synthesize cDNA separately. The cDNAs were used as templates to amplify conotoxin genes by PCR.The optimized RNA isolation and cDNA synthesis methods were obtained according to RT-PCR results, which would be the basis for cloning of new conotoxin genes.

Objective To isolate and clone novel O-superfamily conotoxin genes of Conus capitaneus Linnaeus collected from Hainan,which would provide the initial drug leads to investigate and develop Conus capitaneus conotoxins.Methods 3'-RACE(Rapid Amplification of cDNA Ends) was used for cloning the novel O-superfamily conotoxins.The specific amplified cDNA fragments were sequenced and analyzed,as well as the genetic diversity of the O-superfamily conotoxins.Results The full-length cDNA(CaHr91N) of a new O-superfamily conotoxin(CaHr91) was cloned and sequenced from Conus capitaneus Linnaeus.The novel conotoxin precursor CaHr91P with 77 amino acids(aa) encoded by the cDNA consists of three typical regions of signal with 21aa, pro-peptide with 22aa and mature peptide with 34aa.Predicted sequence of the toxin region CaHr91M is "ECREQSQGC TNTSPPCC SGLRC SGQSQGGVC ISN" with a common O-superfamily cysteine pattern C-C-CC-C-C.Percent identities between CaHr91P and other published homologue O-superfamily sequences were compared systematically,as well as research status on conopeptides from C.capitaneus.Conlusion The elucidated cDNA of the novel CaHr91P conotoxin will be the basis for a better understanding of its bioactivity and application,as well as finding more novel conotoxins from C.capitaneus.

AIM:To compared the differential sensitivity of nicotinic acetycholine receptors (nAChRs) consisting of α and β subunits with different ratios.METHODS:The cRNA of α and β subunits was obtained by in vitro transcription.The α3β2 and α3β4 nAChR subtypes were expressed in Xenopus laevis oocytes by microinjection of cRNA coding α and β subunits at α∶β ratios of 1∶10, 1∶1 and 10∶1.The pharmacological activities of nAChRs to agonist acetycholine (ACh) and antagonist α-conotoxin (CTx) RegⅡA were investigated by two-electrode voltage-clamp techniques.RESULTS:For α3β2 nAChR expressed at the ratios of 1∶10, 1∶1 and 10∶1, the EC50 values of ACh were 91.2 μmol/L, 104.4 μmol/L and 130.6 μmol/L, respectively, while the IC50 values of α-CTx RegⅡA were 40.2 nmol/L, 36.4 nmol/L and 42.3 nmol/L, respectively.For α3β4 nAChR at the ratios of 1∶10, 1∶1 and 10∶1, the EC50 values of ACh were 44.0 μmol/L, 110.0 μmol/L and 230.0 μmol/L, respectively, while the IC50 values of α-CTx RegⅡA were 226.8 nmol/L, 71.5 nmol/L and 49.4 nmol/L, respectively.CONCLUSION:The results imply that the α3 and β4 subunit stoichiometry can change the structure and pharmacological activity of α3β4 nAChR, but the stoichiometry of α3 and β2 subunits has no effect on α3β2 nAChR.

To investigate activities of three isomers of α-conotoxin TxID on human α3β4 and α6/α3β4 nicotinic acetylcholine receptors(nAChRs). The three isomers of α-conotoxin TxID were synthesized using solid phase Fmoc chemistry and fully folded by two-step oxidations. Human α3β4 and α6/α3β4 nAChRs were expressed in oocytes of Xenopus laevis, which were used for bioassay of the three isomers, including inhibition and washout reversibility. There were obvious differences between the inhibition potency of each isomers at human α3β4 and α6/α3β4 nAChRs. The blocking was reversible and washout rapidly. The most potent isomer is the globular form with an IC50 of 9. 3 nmol/L on human α3β4 and α6/α3β4 nAChRs respectively. The 2nd potent isomer was the ribbon form with much less potency, which had an IC50 of > 5 μmol/L. The bead isomer had little or no block on human α3β4 and α6/α3β4 nAChRs with an IC50 of > 10 μmol/L. The three isomers of α-conotoxin TxID were synthesized successfully with two pairs of desired disulfide bond. Inhibition activities of the 3 isomers on human α3β4 and α6/α3β4 nAChRs were obtained respectively, which would be basis for new marine drug development of α-conotoxin TxID.