| 人鸟氨酸脱羧酶抗酶1突变基因的克隆与原核表达 |
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| 引用本文： | 蔡富强,韩钰,王艳林.人鸟氨酸脱羧酶抗酶1突变基因的克隆与原核表达[J].生物技术通报,2010(2). |
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| 作者姓名： | 蔡富强 韩钰 王艳林 |
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| 作者单位： | 1. 三峡大学分子生物学研究所,宜昌,443002
2. 三峡大学分子生物学研究所,宜昌,443002;三峡大学医学院,宜昌,443002 |
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| 基金项目： | 国家自然科学基金资助项目，湖北省教育厅重点研究资助项目 |
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| 摘 要： | 克隆人鸟氨酸脱羧酶抗酶1(Homo sapiensornithine decarboxylase antizyme 1,HOAZ1)开放性阅读框+1核糖体移码位点缺失的突变基因,构建突变基因的原核表达质粒,分离纯化其原核表达的重组蛋白。采用巢式-PCR和重叠延伸-PCR技术,从人非小细胞肺癌细胞株A549的cDNA中获得人类鸟氨酸脱羧酶抗酶1开放性阅读框+1核糖体移码(+1RF)位点缺失突变的基因序列(DM-HOAZ1)。将该序列克隆到原核表达载体pET-28a(+)后,转化表达菌Rosseta(DE3)感受态细胞。阳性克隆用IPTG诱导重组蛋白表达,然后在尿素变性条件下经Ni-NTA树脂亲和层析纯化重组HOAZ1。原核表达和纯化的HOAZ1重组蛋白用Western Blot鉴定。结果显示,成功获得HOAZ1开放阅读框中+1RF位点缺失的突变基因和该突变基因的原核表达质粒pET-28a(+)/DM-HOAZ1;用pET-28a(+)/DM-HOAZ1转化大肠杆菌后,HOAZ-1可被IPTG诱导性高表达,且表达量随诱导时间延长递增;原核表达的HOAZ1可用Ni-NTA树脂亲和层析有效纯化。建立了原核表达和分离纯化HOAZ1蛋白的试验方法,为进一步研究HOAZ1的功能和临床应用奠定了基础。
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| 关 键 词： | 鸟氨酸脱羧酶抗酶1 突变 原核表达 |
Cloning and Prokaryotic Expression of Mutated Human Ornithine Decarboxylase Antizyme 1 |
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| Cai Fuqiang,Han Yu,Wang Yanlin.Cloning and Prokaryotic Expression of Mutated Human Ornithine Decarboxylase Antizyme 1[J].Biotechnology Bulletin,2010(2). |
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| Authors: | Cai Fuqiang Han Yu Wang Yanlin |
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| Affiliation: | Cai Fuqiang 1 Han Yu 1 Wang Yanlin 1,2(1Institute of Molecular Biology,Medical College,Yichang 443002,2 Three Gorges University,Yichang 443002) |
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| Abstract: | It was to clone human ornithine decarboxylase antizyme 1 with deleted-mutation in +1 ribosomal frame shifting(+1RF)site(DM-HOAZ1),and to establish a method for prokaryotic expression and purification of the HOAZ1.Nest-PCR and overlapping extension-PCR were used to amplify DM-HOAZ1 from cDNA of human non-small lung cancer cell line A549.DM-HOAZ1 was then inserted into pET-28a(+)vector to construct plasmid pET-28a(+)/DM-HOAZ1 for prokaryotic expression.pET-28a(+)/DM-HOAZ1 was transformed into E.coli Rosseta(D... |
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| Keywords: | Ornithine decarboxylase antizyme 1 Mutation Prokaryotic expression |
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