Eukaryotic Expression And Function Study Of Toxins From The Venom Of The Spider Chilobrachys Jingzhao

The tarantula Chilobrachys jingzhao is one of the most venomous spiders,found recently in Guangxi and Yunnan in the south of China. The venom of C.jingzhao is a mixture of various components with different types of biological activities.We found two toxin peptides from it,named JZTX-34 and JZTX-â…¢.JZTX-34 is a 35-residue polypeptide and JZTX-â…¢is 36.The two peptides both contain six cysteines cross-linked by three disulfide bonds.In order to study more about them, we decided to get the two peptides by the molecular cloning and expression.We choose the eukaryotic secretion expression system which. includes the vector of pVT102U/αand the Saccharomyces cerevisiae strain S78.The expressed JZTX-34 was able to inhibit tetrodotoxin-sensitive (TTX-S) sodium currents(IC₅₀～85 nM) while having no significant effects on tetrodotoxin-resistant(TTX-R) sodium currents on rat dorsal root ganglion neurons.The inhibition of TTX-S sodium channels was completely reversed by strong depolarization(+120 mV).Toxin treatment altered neither channel activation and inactivation kinetics nor recovery rate from inactivation.However,100 nM JZTX-34 caused a negative shift of steady-state inactivation curve of TTX-S sodium channels by approximately 10 mV.The results indicated that JZTX-34 might inhibit mammalian sensory neuronal sodium channels but the binding sites should not overlay completely.Besides,there is a amidation signal -RK located in the C-terminal of the JZTX-34 precursor.Unfortunately the expressed JZTX-34 had no amidation modification.This might be the reason why the expressed JZTX-â…¢is denatured easily.The expressed JZTX-â…¢had similar biological activity as those of the native toxin isolated from the spider venom.We cloned and expressed the 25 mutants of JZTX-â…¢.And the differences of their yield are remarkable. Further,that we changed the expression factor of HWTX-XI made the yield increased greatly.With these expressions,we found the factors which influence the expression of toxin peptides are complicated and diversified.These experience will contribute to the expression of future.