首页 | 期刊介绍 | 编委会 | 道德声明 | 投稿指南 | 常用下载 | 过刊浏览 | In English
引用本文:
【打印本页】   【HTML】   【下载PDF全文】   查看/发表评论  下载PDF阅读器  关闭
←前一篇|后一篇→ 过刊浏览    高级检索
本文已被:浏览 94次   下载 80 本文二维码信息
码上扫一扫!
分享到: 微信 更多
基于高效液相色谱法的扇贝毒素-2制备方法研究
王玉1,2, 彭吉星2, 吴海燕2, 郭萌萌2, 佟蒙蒙1,3, 谭志军2
1.浙江大学海洋学院海洋生物研究所 舟山 316021;2.中国水产科学研究院黄海水产研究所 青岛 266071;3.舟山市疾病预防控制中心 舟山 316021
摘要:
本文以渐尖鳍藻(Dinophysis acuminata)为实验原料,建立了基于高效液相色谱法(HPLC)的扇贝毒素-2(Pectenotoxin-2,PTX2)的制备方法。研究重点对鳍藻细胞的破碎、PTX2毒素的提取及HPLC纯化等三个步骤的方法进行筛选和优化。结果表明,反复冻融与超声波细胞破碎相结合可获得最大的细胞破碎率和毒素提取率,分别为99.2%和81.5%。采用四种有机试剂(甲醇、丙酮、乙醚、二氯甲烷)及三种固相萃取小柱(Solid Phase Extraction,SPE)(Oasis HLB SPE,Phenomenex Strata-X SPE,Sep-Pak C18 SPE)对PTX2毒素进行提取,其中乙醚-二氯甲烷的提取效率最高,达78.2%。在HPLC对PTX2毒素的纯化实验中发现,流动相的pH会影响毒素组分的分离,其中在碱性流动相(含6.7mmol/L氨水的水溶液和乙腈溶液,pH=10.94)条件下,PTX2的分离效果最佳,响应值最高。实验使用Phenomenex Kinetex C18(4.8mm×250mm,5μm)色谱柱,在上述碱性流动相条件下,分离得到纯度为93.2%的PTX2毒素。本研究选定的藻细胞破碎、PTX2毒素提取、及HPLC纯化方法简单快捷,高效易操作,为从大规模培养的鳍藻细胞中制备高纯度PTX2毒素提供了技术支持。
关键词:  扇贝毒素-2  鳍藻  高效液相色谱  萃取  制备
DOI:10.11693/hyhz20171000272
分类号:O657.63
基金项目:科技部国家重点研发计划项目,2016YFC1402104号;山东省重点研发计划项目,2016GSF120018号;浙江省海产品健康危害因素关键技术研究重点实验室开放基金资助项目,201606号。
PREPARATION OF PECTENOTOXIN-2 BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY
WANG Yu1,2, PENG Ji-Xing2, WU Hai-Yan2, GUO Meng-Meng2, TONG Meng-Meng1,3, TAN Zhi-Jun2
1.Institute of Marine Biology, Ocean College, Zhejiang University, Zhoushan 316021, China;2.Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China;3.Key Laboratory of Testing and Evaluation for Aquatic Product Safety and Quality, Ministry of Agriculture, Zhoushan 316021, China
Abstract:
A protocol for preparing pectenotoxin-2 (PTX2) from a large-scale culture of Dinophsis acuminata was conducted in high-performance liquid chromatography (HPLC). Methods of cell disruption, toxin extraction, and HPLC purification were selected and optimized. The results indicate that the most efficient cell disruption and toxin extraction were achieved in the combination of freezing + thawing + ultrasound, resulting in 99.2% and 81.5%, respectively. PTX2 was extracted using liquid-liquid extraction (methanol, acetone, diethyl ether, and dichloromethane) and Solid Phase Extraction columns (Oasis HLB SPE, Phenomenex Strata-X SPE, Sep-Pak C18 SPE). PTX2 extraction efficiency reached maximum (78.19%) with diethyl ether and dichloromethane. Studies on the optimization conditions of HPLC showed that the pH value of the mobile phase had an impact on the chromatographic reaction. When mobile phase was in alkaline condition (containing 6.7mmol/L ammonium hydroxide in acetonitrile/water, pH=10.94), PTX2 could be better chromatographically separated by Phenomenex Kinetex C18 (4.8mm×250mm, 5μm). The final PTX2 reached 93.2% in purity. This method was rapid, effective, and comprehensive for extraction, separation, and purification of PTX2 from D. acuminata cells with HPLC.
Key words:  pectenotoxin-2  Dinophysis acuminata  high-performance liquid chromatography  extraction  preparation
版权所有 《海洋与湖沼》编辑部 Copyright©2008 All Rights Reserved
主管单位:中国科学技术协会 主办单位:中国海洋湖沼学会 中国科学院海洋研究所
地址:青岛市南海路七号  邮编:266071  电话:0532-82898753  E-mail:liuxiujuan@qdio.ac.cn
技术支持:北京勤云科技发展有限公司