| SDH全基因的拼接及原核表达 |
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| 引用本文： | 涂继方,汪旭珍,徐莉. SDH全基因的拼接及原核表达[J]. 生物技术, 2009, 19(2) |
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| 作者姓名： | 涂继方 汪旭珍 徐莉 |
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| 作者单位： | 浙江中医药大学,浙江杭州,310053 |
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| 基金项目： | 浙江省医药卫生科学研究基金，浙江省自然科学基金，浙江省教育厅科学研究计划项目 |
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| 摘 要： | 目的:构建闭联异松树脂醇二脂脱氢酶(SDH)全基因片段与原核表达质粒载体,在大肠杆菌中进行表达.方法:将SDH3'端和5'端序列通过PCR方法拼接获得全基因后,然后将其插入到相应的原核表达质粒载体PGEX-6p-1中,形成了重组载体,并使其在大肠杆菌BL21中经IFFG诱导其表达蛋白,再用SDS-PAGE电泳检测表达结果.结果:成功的将SDH基因片段拼接成全基因,并将其构建入原核表达载体PGEX-6p-1中.测序结果表明载体构建成功,无插入和移码突变.经1.5mmol/LIPTC诱导后获得与预测大小(29.253kDa)完全一致的目的蛋白即闭联异松树脂醇二脂脱氢酶蛋白,并测得在37℃、250r/min的实验条件下的最佳诱导时间为4h.结论:成功获得了重组质粒并在大肠杆菌中较好的诱导表达得到了闭联异松树脂醇二脂脱氢酶的蛋白.
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| 关 键 词： | 原核表达 拼接 原核载体 闭联异松树脂醇二脂脱氢酶 |
The Entire Gene SDH Splicing and Prokaryotic Expression |
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| TU JI-fang,WANG Xu-zhen,XU Li. The Entire Gene SDH Splicing and Prokaryotic Expression[J]. Biotechnology, 2009, 19(2) |
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| Authors: | TU JI-fang WANG Xu-zhen XU Li |
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| Affiliation: | TU JI-fang,WANG Xu-zhen,XU Li (Zhejiang Chinese Medical University,Hangzhou 310053,China) |
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| Abstract: | Objective:Obtain the entire gene Secoisolariciresinol Dehydrogenase(SDH)and constructed it into the IPTG inducible prokaryotic expression plasmid (PGEX-6P-1) in E.coli. Method: Obtaining the entire SDH gene fragment by PCR splicing, then amplify the entire SDH gene sequence by PCR, insert it into the prokaryotic express vector PGEX-6p-1.Then the recombinant plasmid was transferred into E.coli BL21 and induced with IPTG. Finally, the results was detected by SDS-PAGE. Result: The SDH gene was amplified and th... |
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| Keywords: | prokaryotic expression splicing prokaryotic vector secoisolariciresinol dehydrogenase |
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