Abstract: A new T-superfamily conotoxin B8.1T with sequence DCCPESPPCCH was discovered from C. betulinus native to Hainan. Conotoxin B8.1T contains two disulphide bridges between Cys3-Cys9, Cys4-Cys10. The linear conotoxin B8.1T was synthesized by Fmoc solid-phase synthesis method at first. Three oxidative methods were used for folding linear peptides according to different cysteine thiol protecting groups：（1）two-steps oxidative folding；（2）one-pot folding；（3）random one-step oxidative folding. Oxidative folding parameters were optimized to improve folding efficiency. The bioactivity of conotoxin B8.1T was assayed. The results demonstrated that all the three strategies can be used to produce native conotoxin B8.1T. However, the efficiencies of the two-step and one-pot methods were higher than that of the one-step. The presence of folding additives helped to improve native isomer accumulating by parameters’ optimization. The bioassay showed that conotoxin B8.1T inhibited the movement behavior of mice by intracranial injections, while there was no effects on goldfish by intramuscular injections. The results would lay a solid foundation for follow-up synthesis and research of T-superfamily contoxins.

Key words: T-superfamily conotoxin, Solid peptide synthesis, Oxidative folding, Disulfide bond formation