虎纹捕鸟蛛毒素-XVⅡa的基因克隆及在酿酒酵母中的表达

Gene cloning and expression of Huwen toxin XVⅡa from the spider Ornithoctonus huwena

目的基因克隆和表达虎纹捕鸟蛛毒素-XVⅡa,并进行纯化和质谱鉴定。方法通过随机测序虎纹捕鸟蛛(Ornithoctonus huwena)毒腺cDNA文库,获得编码多肽毒素HWTX-XVⅡa的基因,对其进行生物信息学分析;构建HWTX-XVⅡa基因真核表达质粒,利用S78株酿酒酵母(Saccharomyces cerevisiae)表达系统进行分泌表达,表达产物经离子交换、反相高效液相色谱分离纯化和质谱鉴定。结果克隆得到虎纹捕鸟蛛毒素-XVⅡa的基因,构建的真核表达质粒表达产物纯化后进行质谱鉴定,为分子质量单位为3.250 004ku的虎纹捕鸟蛛毒素-XVⅡa。结论获得虎纹捕鸟蛛毒素-XVⅡa的基因及其表达产物,为研究其生物学功能奠定了基础。

Objective To clone and express the peptide of Huwen toxin XVⅡa（HWTX-XVⅡa）from the spider Ornithoctonus huwena. Methods Random clones were sequenced from a library of cDNA from the venom gland of the spider O.huwena.A novel gene encoding HWTX-XVⅡa was cloned and was analyzed using bioinformatics.An expression plasmid was constructed and expressed in the S78 strain of Saccharomyces cerevisiae.The product of expression was purified using reversed phase liquid chromatography and identified using MALDI-TOF mass spectrometry. Results A novel gene,named HWTX-XVⅡa,was cloned into a recombinant plasmid that was transformed into S.cerevisiae S78.The molecular mass of the expressed peptide was 3.25 ku. Conclusion The novel gene HWTX-XVⅡa was expressed in S.cerevisiae to lay the foundation for further research into its biological functions.