Gene Cloning And Expression Of Toxins From Two Spiders

BackgroundNowadays, sciencetists on natural drug are paying more and more attention to spider toxins and their physical functions, which are believed to be potential drug molecules. However, the yield of natural venoms limits the study of the toxins’function. A variety of proteins and peptides certificated to be with biological activity are identified from the venom of Ornithoctonus huwena, which is widely distributed in hilly areas in southern China. Peptides, called DkTx （double-knot toxin）, can activate the capsaicin receptor and transient receptor potential vanilloid subtype1（TRPV1） channel to promote the release of calcitonin-related peptide with releasing vasculars and lowering blood pressure, by targeting cell membrane outer pore domain. Agelena silvatica is widely distributed in the mountains and steppe regions in China. It can capture preys by paralysising them with venom. At present, there is little study about the venom of Agelena silvatica, so my investigation would lay basis for other studys on the venom.Objective1To screen DkTx-like peptides, and to analysis the biological function.2To construct the cDNA library of Agelena silvatica venom gland, and to screen new toxin genes.Methods1Amplifying the target genes by PCR with specific primers, and constructing the target genes to expression vector pVT102U/a, to express the peptides with Saccharomyces cerevisiae strain S-78.2Screening new toxin genes by PCR with Agelena silvatica venom gland cDNA library, which was constructed with CreatorTM SMARTTM cDNA Library Construction Kit.Result1Four DkTx-like peptides, FR+T3F34+P-5, FR+T3F34+P-6, FR+T5R32+P-8, FR+T5R32+P-105, were obtained in Ornithoctonus huwena venom gland cDNA library. And some were expressed with Saccharomyces cerevisiae strain S-782Five cDNA sequences, D7, D82, D134, D523, D787, were screened in Agelena silvatica venom gland cDNA library. And D7, D134, D523, D787were identified to be new spider venom genes. D82is same with a venom gene in super family Toxin₇of Agelenopsis aperta.Conclusion1Four DkTx-like peptides were screened and expressed artificially.2Four new spider venom genes were cloned from Agelena silvatica venom gland cDNA library.