少棘蜈蚣钾通道毒素多肽KTX-Ssm175的表达、纯化与鉴定  

Expression,Purification and Identification of Potassium Channel Toxins KTX-Ssm175 from Scoropendra subspinipes mutilans

作　　者：尹世金[1] 陈璇[1] 闻闫瀚 陆春兰[2] 胡青兰[1] 邹艳[1] 张丰[1] 王冠[1] 黄鑫[1] 黄谨[1] 

机构地区：[1]中南民族大学药学院,武汉430074 [2]中南民族大学生物医学工程学院,武汉430074

出　　处：《中南民族大学学报（自然科学版）》2016年第4期43-47,共5页Journal of South-Central University for Nationalities：Natural Science Edition

基　　金：国家自然科学基金资助项目(81373379);湖北省自然科学基金资助项目(2015CFB491);国家级大学生创新训练项目(GCX15009)

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.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 K-SLPTX-Ssml a, suggesting KTX-Ssm175 polypeptide might be a novel voltage-gated potassium channel blocker. A prokaryotic expression vector pGEX-4T-1-KTX-Ssm175 was constructed by genetic engineering and KTX-SsmI75 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 KTX- Ssm175 polypeptide was successfully achieved, providing the material foundation for further study about its structure and function.

关 键 词：少棘蜈蚣 转录组 KTX-Ssm175 钾通道阻断剂 基因工程 

分 类 号：Q812[生物学—生物工程]