Cdna Cloning Of Spider Peptide Toxins And The Toxinomics Study Of The Venom From The Spider Ornithoctonus Huwena Wang

The venom of the spider contain a variety of venomous components with different biological activities. In previous work, the sixteen cDNA sequences from Ornithoctonus huwena Wang and Chilobrachis jingzhao were cloned and sequenced. In this study, five cDNAs encoding five toxins Huwentoxin-XII, Huwen -toxin-XIII, Jingzhaotoxin-tin, Jingzhaotoxin-X and Hainana -toxin-VII were cloned and sequenced by RACE (Rapid amplify -cation of cDNA ends). On the basis of their amino acid sequences, we designed and synthesized 3' RACE and 5' RACE primers. By overlapping the two partial cDNA sequences obtained by 3' and 5' RACE, their full-length sequence were obtained.Compared with the cDNA sequences of Huwentoxin~I, IIIa, -III , IV, and Selenocosmia huwenlectin-I, the prepro region of Huwentoxin-XII was demonstrated to have high similarity (>69%) with these toxins, so we classified Huwentoxin-XII into the superfamily of the former five toxins. However, the prepro region of Huwentoxin-XIII was demonstrated to have low simi -larity with any other from the venom of the three superfamilies found in the spider 0. huwena Wang. Therefore, we infer that Huwentoxin-XIII belong to a novel superfaminly of the venom of the spider 0. huwena Wang , differing from the three super -families in the previous discovery.The cDNA sequences of Jingzhaotoxin-tin and Jingzhaotoxin -X have the same structures with any other JZTX cloned in previous work, the precursors of which composed of a signal peptide, a mature peptide and an intervening sequence. But the length of their propre region are obvious different. The additional amino acid residuces (Gly-Arg-Arg) in the C terminal of the precursors indicate that the carboxyl terminal of Jingzhaotoxin-tin was amidated. Hainanatoxin-Ⅶ, the toxin which had an effect on sodium channel, was purified from Selenocosmia hainana. The cDNA sequences of Hainanatoxin-Ⅶindicates that the C terminal of the precursors has additional amino acid residuces (Phe-Arg -Lys), instead of the signal of amidation.The venomomics research for the bird spider Ornithoctonus huwena Wang was described by the separation and characteri -zation of the crude venom from this spider. In this study, the component with the molecular weight less than 10 kDa were separated by multidimensional chromatography. Firstly, the crude venom of O. huwena Wang was isolated by Gel filtration, and it was divided into fractions containing small proteins (MW＜10 kDa) and fractions containing large proteins (MW＜10 kDa). Then the Fraction containing small proteins (MW＜10 kDa) was isolated and purified by subjected to ion-exchange HPLC and reverse phase HPLC. By using MALDI-TOF MS, we obtained the peptide Mass finger printing of peptide toxins (MW＜10 kDa) from the spider venom of O. huwena gang, which contain about 110 different molecular species. The general scopes for the molecular weight of these components rang from m/z approximate 1500 Da to 8700 Da. Finally, the full amino acid sequences or partial N-terminal sequences of 26 peptides with high purity were determined by Edman-degradation, and their biological activities were characterized.