东亚钳蝎神经毒素基因BmKCT的克隆与表达

Cloning and expression of a neurotoxin from chinese scorpion Buthus martensii Karsch in Escherichia coli

从蝎毒腺中提取总RNA,通过RT-PCR得到东亚钳蝎神经毒素BmKCT的cDNA,将其连接到pUCm-T载体中。序列分析表明,该基因开放阅读框架编码59个氨基酸残基,其中24个为信号肽,其余35个为成熟肽,与Genebank上登录的相同。将蝎神经毒素cDNA再经PCR扩增除去信号肽序列,克隆到pTrcHisA表达载体中,转化大肠杆菌BL21(DE3),经IPTG诱导可高效表达分子质量为10ku左右的融合蛋白。表达产物约占菌体总蛋白的25%。

A neurotoxin cDNA was cloned from Chinese scorpion Buthus martensii Karsch by RTPCR.The cDNA was cloned into the pUCmT vector and sequenced.It has a ORF encoding 59 amino acid residues and a 24 residues signal peptide.The cDNA encoding the mature peptide was amplified by PCR and was cloned into pTrcHisA vector.The recombinant vector was transformed into E.coli BL21(DE3).The E.coli highly expressed the fusion protein whose mollecular weight is 10 ku,after induced by \{0.1 mol/L\} IPTG.The expressed protein was accumulated up to more than 25% of total bacterial protein.