东亚钳蝎K+通道阻断肽cDNA的克隆与表达

目的：克隆东亚钳蝎毒素基因，以进一步研究其生物学和药理学功能。方法：利用已知蝎神经毒素基因序列，设计引物，用RT-PCR方法克隆从蝎毒腺组织蝎毒素cDNA。结果：成功地克隆了一个新的东亚钳蝎毒素基因，该基因开放阅读框架编码59个氨基酸残基，其中前22个为信号肽，成熟肽为37个氨基酸残基，经PCR扩增除去信号肽序列，克隆到pTreHisA质粒中，在E．coli中表达了分子质量为7ku左右融合蛋白，表达产物占菌体总蛋白的21％左右。结论：其结构中含有三对二硫链，6个Cys残基组成蝎K^ 通道毒素共同特征序列-CXXXC-、-GXC-、-CXC-，推断其为K^ 通道阻断肽，命名为KChTX1。已被Gene-bank收录，收录号为AY129234。

Cloning and Expression of a K+ Channel- blocking Neurotoxin cDNA from Chinese scorpion Buthus Martensii Karsch

A K~ channel-blocking gene 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 22 residues signal peptide.It contain a structure of -CXXXC-,-GXC-,-CXC- that the common characteristic of scorpion K~ channel-blocking peptide.so,To make a conjecture that the cDNA encoding a K~ channel-blocking peptide,and named it is KChTX1.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 inclusion body whose mollecular weight is 7 ku.The expressed protein was accumulated up to more than 21 % of total bacterial protein.