| 刺参甘露糖结合凝集素的原核表达、纯化及生物活性分析 |
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| 引用本文： | 李丹彤,谢广成,丁文勇,王秀利,刘洋,徐文琦,张永攀.刺参甘露糖结合凝集素的原核表达、纯化及生物活性分析[J].水产学报,2011,35(8):1166-1171. |
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| 作者姓名： | 李丹彤 谢广成 丁文勇 王秀利 刘洋 徐文琦 张永攀 |
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| 作者单位： | 1. 大连海洋大学农业部海洋水产增养殖学重点开放实验室,辽宁大连 116023;大连海洋大学辽宁省海洋生物资源与生境修复重点实验室,辽宁大连 116023
2. 大连医科大学生物化学教研室,辽宁大连,116044 |
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| 基金项目： | 辽宁省教育厅团队项目(2008T021) |
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| 摘 要： | 为进一步研究刺参甘露糖结合凝集素(AJ-MBL)的功能及提高刺参自身免疫力,对AJ-MBL基因的CDS区进行原核表达、纯化并初步鉴定其生物学活性。采用RT-PCR法扩增AJ-MBL基因编码区,经酶切鉴定、序列分析后,将其CDS区498 bp片段克隆至原核表达载体pET28a中,得到重组质粒pET-AJ-MBL。重组质粒转化大肠杆菌BL21(DE3)感受态细胞并经IPTG诱导表达出分子量约17 ku的融合蛋白。该蛋白以包涵体形式存在,用Ni2+离子亲和柱纯化。结果显示,获得高表达量的重组纯化蛋白。纯化的17 ku AJ-MBL进行红细胞凝集试验检测其生物学活性。凝集试验结果显示,AJ-MBL凝集最小浓度为10 μg/mL。本实验成功构建了AJ-MBL原核表达质粒,并在大肠杆菌中高效表达了17 ku AJ-MBL蛋白,该蛋白具有良好的生物学活性。
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| 关 键 词： | 刺参 甘露糖结合凝集素 原核表达 蛋白纯化 红细胞凝集试验 |
| 收稿时间： | 1/5/2011 9:39:02 AM |
| 修稿时间： | 5/4/2011 9:50:51 PM |
Prokaryotic expression,purification and bioactivity analysis of Apostichopus japonicus mannan binding lectin |
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| LI Dan-tong,XIE Guang-cheng,DING Wen-yong,WANG Xiu-li,LIU Yang,XU Wen-qi and ZHANG Yong-pan.Prokaryotic expression,purification and bioactivity analysis of Apostichopus japonicus mannan binding lectin[J].Journal of Fisheries of China,2011,35(8):1166-1171. |
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| Authors: | LI Dan-tong XIE Guang-cheng DING Wen-yong WANG Xiu-li LIU Yang XU Wen-qi and ZHANG Yong-pan |
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| Institution: | dalian ocean university,dalian ocean university,Dalian Medical University,dalian ocean university,dalian ocean university,dalian ocean university,dalian ocean university |
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| Abstract: | To further study the functional characteristics of Apostichopus japonicus mannan-binding lectin(AJ-MBL)and to improve the natural immunity of A.japonicus,a prokaryotic vector of AJ-MBL gene CDS region was constructed.The fusion protein was expressed and purified in prokaryotic system and its bioactivity was studied.The coding sequence of AJ-MBL was amplified by RT-PCR method.After being identified by the restriction digestion and sequencing,the 498 bp of AJ-MBL gene was inserted into pET28a plasmid to yield... |
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| Keywords: | Apostichopus japonicus mannan-binding lectin prokaryotic expression purification hemagglutination assay |
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