| 人Delta-like4~(ext)-93-217原核表达、纯化及蛋白活性检测 |
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| 引用本文： | 伍艳兰,黄斯勇,牛晓丽,张丽,陈茹菲,何飞,张萍,梁英民,刘利.人Delta-like4~(ext)-93-217原核表达、纯化及蛋白活性检测[J].第四军医大学学报,2009(17). |
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| 作者姓名： | 伍艳兰 黄斯勇 牛晓丽 张丽 陈茹菲 何飞 张萍 梁英民 刘利 |
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| 作者单位： | 第四军医大学唐都医院血液科;第四军医大学基础部医学遗传学与发育生物学教研室; |
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| 基金项目： | “十一五”国家“863”重大、重点项目(2006AA02A111) |
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| 摘 要： | 目的:原核表达、纯化融合蛋白TRX/hDll4ext-93-217,并对其活性进行检测.方法:采用PCR方法扩增hDll4ext-93-217多核苷酸序列,克隆入pMD18T载体.测序正确后,将其亚克隆入pET32a(+)原核表达载体,获得pET32a(+)-hDll4ext-93-217载体.以该载体转化E.coli菌株BL21,IPTG诱导其表达.以镍离子螯合柱纯化目的蛋白,采用SDS-PAGE,Western Blot方法鉴定目的蛋白的表达;采用萤光素酶报告基因系统检测目的蛋白活性.结果:通过PCR扩增获得了目的片段hDll4ext-93-217,成功构建了该蛋白的原核表达载体pET32a(+)-hDll4ext-93-217.采用SDS-PAGE,Western Blot等方法均可检测到目的可溶性融合蛋白TRX/hDll4ext-93-217的表达.融合蛋白TRX/hDll4ext-93-217在25℃,IPTG终浓度0.5mmol/L条件下诱导表达8h的蛋白表达量最高.以镍离子螯合柱纯化,获得纯化融合蛋白TRX/hDll4ext-93-217浓度为1.5g/L,其荧光素酶报告基因的刺激活性与空白对照...
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| 关 键 词： | Detla-like4 Notch信号途径 造血干细胞 原核表达 |
Prokaryotic expression,purification and protein activity detection of human Delta-like4~(ext)-93-217 |
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| WU Yan-Lan,HUANG Si-Yong,NIU Xiao-Li,ZHANG Li,CHEN Ru-Fei,HE Fei,ZHANG Ping,LIANG Ying-Min,LIU Li.Prokaryotic expression,purification and protein activity detection of human Delta-like4~(ext)-93-217[J].Journal of the Fourth Military Medical University,2009(17). |
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| Authors: | WU Yan-Lan HUANG Si-Yong NIU Xiao-Li ZHANG Li CHEN Ru-Fei HE Fei ZHANG Ping LIANG Ying-Min LIU Li |
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| Institution: | WU Yan-Lan1,HUANG Si-Yong1,NIU Xiao-Li1,ZHANG Li1,CHEN Ru-Fei1,HE Fei2,ZHANG Ping2,LIANG Ying-Min1,LIU Li1 1Department of Hematology,Tangdu Hospital,Fourth Military Medical University,Xi'an 710038,China,2Department of Medical Genetics and Developmental Biology,Xi'an 710033 |
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| Abstract: | AIM:To express and purify the fusion protein TRX/hDll4ext-93-217 and to detect its protein activity.METHODS:The cDNA sequence of hDll4ext-93-217 was amplified by PCR and cloned into the vector pMD18T.After sequencing,the correct fragment was cloned into prokaryotic expression plasmid pET32a(+).The target protein was expressed in E.coli BL21 induced by IPTG and the expression was detected by SDS-PAGE and Western blotting.The activity of purified target protein with NI-NTA was detected by luciferase report as... |
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| Keywords: | Detla-like4 Notch signaling pathway hematopoietic stem cells prokaryotic expression |
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