Gene Cloning, Expression And Structure-Function Study Of Toxins From The Spider Selenocosmia Huwena Wang [=Ornithoctonus Huwena (Wang)]

The spider Selenocosmia huwena Wang[=Ornithoctonus huwena (Wang)] is distributed in the hilly areas of Guangxi and Yunnan in the south of China. In previous work, many components with different structures and functions were purified and characterized. The expression of spider toxins by genetic engineering often encountered difficulties for the pairs of disulfide bonds, short-chain peptide and other unknown reasons. In this study, we tried to express toxins from the spider of S. huwena Wang in different paths such as prokaryotic secretion expression and eukaryotic secretion expression. First, the synthetic HWTX-IV gene was expressed in the eukaryotic secretion expression vector of pPIC9K, but unfortunately, we can' t purify the recombinant HWTX-IV. Then the gene of HWTX-IV has been subcloned into the prokaryotic vector pMAL-p2X. The fusion protein, whose N terminus encodes the Maltose Binding Protein, was expressed by IPTG induction. After cold osmotic shock, the fusion protein was cleaved with thrombin. And the recombinant HWTX-IV was purified by size exclusion chromatography and reversed phase HPLC. The purified rHWTX-IV was proved to be correctly expressed by mass spectrum. The recombinant HWTX-IV was proved to have the same biological activity as the native one.Secondly, HWTX-XI, the first serine proteinase inhibitor isolated from spider venoms which belongs to the BPTI/Kunitz type serine proteinase inhibitor group, was obtained using the vector of pMAL-p2X, whose yield is 0.8mg/L, was proved to be correctly expressed by massspectrum. The recombinant HWTX-XI was proved to have the same inhibition activity as the native one. Then we got a new genetic engineering pathway to express spider toxins and this will benefit the whole spider toxin research.Finally, for the sake of the structure and function of HWTX-XI, some mutants of HWTX-XI were expressed and purified using the eukaryotic secretion expression vector pVT102U. The mutants were analyzed using photospectrometry method and surface plasmon resonance assay. The analysis shows that the mutant K14A' s inhibition level has declined 3107. 5 times. So the main active site of HWTX-XI as a trypsin inhibitor was K14. and the effects of some other side position mutations were also analyzed using photospectrometry method and surface plasmon resonance assay.