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采用Illumina II代转录组测序技术构建了湖北少棘蜈蚣毒腺转录组数据库,从中筛选到一个全新的毒素多肽编码基因KTX-Ssm175.经序列比对显示KTX-Ssm175多肽与κ-SLPTX-Ssm1a高度同源,提示KTX-Ssm175多肽为一种新的电压门控性钾通道阻断剂.采用基因工程技术构建了p GEX-4T-1-KTX-Ssm175原核表达载体,通过GST融合蛋白表达法对KTX-Ssm175多肽在大肠杆菌Rosetta中进行诱导表达和分离纯化.MALDI-TOF-MS质谱测定显示纯化的KTX-Ssm175多肽分子量与理论值相吻合,说明成功实现了KTX-Ssm175多肽的基因工程制备,为深入研究其结构与功能提供了物质基础.
The Illumina II transcriptome sequencing technology was used to construct a database of the genome of the spiked gynoecium of the Chinese spiny centipede from Hubei Province, and a new toxin polypeptide coding gene KTX-Ssm175 was screened from it. The sequence alignment showed that the KTX-Ssm175 polypeptide was highly homologous to the kappa-SLPTX-Ssm1a Source, suggesting that KTX-Ssm175 polypeptide is a new voltage-gated potassium channel blocker.The prokaryotic expression vector pGEX-4T-1-KTX-Ssm175 was constructed by genetic engineering and expressed by GST fusion protein, Ssm175 polypeptide was induced and expressed in E.coli Rosetta and purified by MALDI-TOF-MS.The molecular weight of the purified KTX-Ssm175 polypeptide was consistent with the theoretical value, indicating that the genetic engineering of KTX-Ssm175 polypeptide was successfully achieved, In-depth study of its structure and function provides the material basis.