一种富含半胱氨酸的蜘蛛多肽基因的克隆和初步表达

Gene cloning and expression of a cysteine-rich peptide from the spider Orinithoctonus huwena

目的克隆编码虎纹捕鸟蛛多肽的基因并表达蜘蛛多肽,为研究其生物学功能奠定基础。方法通过构建和随机测序毒腺cDNA文库,克隆编码毒素多肽的基因;构建真核表达质粒,利用酿酒酵母S78株表达系统分泌表达,表达产物进行Tricine SDS-PAGE鉴定。结果克隆到一种编码富含半胱氨酸的多肽的新基因,命名为HWTX-XIVa2基因;构建的真核表达质粒转化酵母S78后表达6.5ku的多肽。结论构建并筛选毒腺cDNA文库是发现毒素多肽基因的一种有效方法,利用基因工程表达是获得蜘蛛多肽的一种有效手段。

Objective To clone and express peptides from the spider Ornithoctonus huwena and to lay the foundation for further research into their biological functions.Methods A venom gland cDNA library was constructed for the spider O.huwena and random clones were sequenced.Genes encoding peptides were cloned,eukaryotic expression plasmids were constructed,and Saccharomyces cerevisiae strain S78 was used as a secretory expression system.The expression yield was identified by Tricine SDS-PAGE.Results A novel gene,named HWTX-XIVa2,encoding a cysteine-rich peptide was cloned.The recombinant plasmids were transformed into S.cerevisiae S78.The molecular mass of the expressed peptide was about 6.5 ku.Conclusion The construction and screening of the venom gland cDNAs library is a promising method with regard to the efficient characterization of potential peptide toxins.Expression in S.cerevisiae S78 is a reliable method of obtaining peptides.