Study On Isolation, Purification, Toxicity Assay, Gene Cloning And Prokaryotic Expression Of Stonefish Toxin From South China Sea

Stonefish are the most venomous and dangerous fish in the world and inhabit inshallow waters of the tropical or subtropical Indo-Pacific region. Their venom cancause a variety of symptoms, such as intense pain, edema, muscle paralysis andhypotension. Toxin neoVTX has been isolated from stonefish venom. The stonefishSynanceia verrucosa are mainly distributed in South China Sea.Object: In order to probe the composition and toxicity of stonefish toxin from SouthSea and prepare its antibody for the treatment of envenomation.Method：Extracted stonefish venom was identified by SDS-PAGE and MS. Gelfiltration HPLC and AKTA protein purification system were used to isolate proteincomponents in small and large amount. RNA was extracted from venomous sacs andgenes of toxins were cloned by RACE. cDNA of toxins was ligated into pET-22b(+)vector and expressed in E.coli.Results:1.2reported and4new proteins in stonefish venom were observed in theelectrophoresis.2proteins were identified as α-and β-subunit of neoVTX. Amongthe4new proteins,2proteins had high matching degree with neoVTX β. Theother2had high matching degree with β tubulin and glyceraldehyde-3-phosphatedehydrogenase, respectively.2. GF-HPLC of venom indicated that there were5unreported proteins whosemolecular weight was about14KD.3. LD50（i.v., to mouse）of crude venom was106ng/g and LD50（i.v., to mouse）ofneoVTX was66ng/g. Proteins whose molecular weight was about14KD causedno toxic symptoms in mice at278ng/g. 4. cDNA sequences of neoVTX α and β were cloned and they had as high as99%homology with neoVTX α and β of Synanceia verrucosa from sea of Okinawa,Japan.5. neoVTX α and β were expressed in pET-22b(+) vector in Rosetta competent cells.neoVTX α was yielded with a productivity of0.45mg/L while neoVTX β subunitwas not expressed efficiently.Conclusions:1. Besides neoVTX, there were at least3kinds of proteins in the venom ofSynanceia verrucosa. The first class of proteins matched highly with neoVTX βsubunits and was inferred as the toxins with lower molecular weight or thedegradation fragments of neoVTX α and β subunits. The second one was inferredas structural proteins and metabolic enzymes. The third one was proteins whosemolecular weight was about14KD.2. Proteins whose molecular weight was about14KD displayed no toxicity,suggesting they were not toxins.3. Expression craft of neoVTX α was explored for the first time.