【摘要】 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.更多还原

【Abstract】 A new toxin polypeptide gene of KTX-Ssm175 was screened by constructing the venomous transcriptome database of Scolopendra subspinipes mutilans from Hubei Province,using Illumina second generation transcriptome sequencing technology. Sequence alignment showed that the polypeptide of KTX-Ssm175 was highly homologous to κ-SLPTX-Ssm1 a,suggesting KTX-Ssm175 polypeptide might be a novel voltage-gated potassium channel blocker. A prokaryotic expression vector p GEX-4T-1-KTX-Ssm175 was constructed by genetic engineering and KTX-Ssm175 polypeptides were expressed and purified in E. coli Rosetta by GST fusion protein expression. MALDI-TOF-MS mass spectrometry showed that the molecular weight of the purified KTX-Ssm175 were consistent with the theoretical value. So genetic engineering preparation of KTXSsm175 polypeptide was successfully achieved,providing the material foundation for further study about its structure and function.更多还原