The Molecular Diversity And Bioactivity Of Toxins From The Spider Cyriopagopus Longipes

Cyriopagopus longipes is a large tarantula inhabiting Southeast Asian countries such as Thailand,Laos and Cambodia.Up to now,there is no report on the study of toxin from Cyriopagopus longipes.This paper will study the diversity of peptide toxin and venom activity of Cyriopagopus longipes for the first time,and lay a foundation for further study of peptidetoxinofCyriopagopuslongipes.Firstly,we constructed a DNA Library of the venom gland of the spider,randomly selected the clones from the library and sequenced them,and obtained 323 expression sequence tags.These expressed sequence tags were analyzed by software.120 of them encoded toxin-like sequences,46 of them encoded sequence that be relevant to cellular transcripts and 157 of them failed to match any known sequence.The software predicted 323 expressed sequence tags,and showed that these expressed sequence tags could express 104 toxin precursor sequences.After further comparing the sequence similarities of these toxin precursor sequences,they were classified into 10 superfamilies.By comparing the sequences of 10 superfamilies,it was found that most of the peptide toxins contained three disulfide bonds,a small part had four disulfide bonds,and very few toxins were linear peptides without disulfide bonds.Most precursor sequences of Cyriopagopus longipes was similar to that of Ornithoctonus huwena.By constructing phylogenetic tree and comparing sequences,we analyzed the sequence differences of toxin precursors of the two spiders,and found that some of the toxin precursors of the two spiders had high similarity and high affinity in evolutionary relationship,which provided a basis for studying the evolutionary relationship between the two spiders.Isolated the crude venom of this spider by high efficiency liquid chromatography and obtained around 40 chromatography peaks.The result of mass spectrometry show that these chromatography peaks consist of 64 peptides and more than half of the peptides were in the range of 3000-5000Da.Animal experiments showed that the LD₅₀of the crude venom was 0.708mg/kg in mice,and the lethal effect of the crude venom on cockroach was weak.The concentration of 200?g/g could only cause paralysis.Patch clamp experiments were conducted to examine the effects of crude venom of Cyriopagopus longipes on voltage-gated ion channels in DRG cells of rats at 100?g/mL.The results showed that crude venom inhibited voltage-gated sodiumchannels,voltage-gatedpotassiumchannels,voltage-activated calcium channels in DRG cells to a certain extent.The inhibition rate of high voltage-activated calcium channels was the highest,reaching 67.7%+5.1%.