| 小鼠鸟氨酸脱羧酶抗酶1功能基因的克隆及原核表达 |
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| 引用本文： | 刘梦瑶,韩钰,蔡富强,何玲,王艳林.小鼠鸟氨酸脱羧酶抗酶1功能基因的克隆及原核表达[J].粉末涂料与涂装,2010,23(2). |
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| 作者姓名： | 刘梦瑶 韩钰 蔡富强 何玲 王艳林 |
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| 作者单位： | 三峡大学分子生物学研究所,湖北宜昌,443002 |
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| 摘 要： | 目的克隆小鼠鸟氨酸脱羧酶抗酶1(OAZ1)功能基因,原核表达并纯化OAZ1重组蛋白。方法采用RT-PCR法从小鼠黑色素瘤细胞总RNA中扩增OAZ1基因,通过重叠延伸PCR技术构建无需移码即可全长翻译的功能基因,并构建重组表达质粒pET15b/OAZ1-T,转化大肠杆菌BL21(DE3),经IPTG诱导表达。表达的重组蛋白经Ni2+-NTA亲和层析纯化后,进行SDS-PAGE和Western blot分析。结果重叠PCR法扩增出692bp的OAZ1功能基因,重组表达质粒经酶切及测序鉴定正确,重组蛋白可在大肠杆菌中以包涵体形式高效表达。纯化的重组蛋白纯度可达79.96%,且可与抗His标签抗体特异性结合。结论已成功克隆了小鼠OAZ1功能基因,原核表达并纯化了重组OAZ1蛋白。
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| 关 键 词： | 鸟氨酸脱羧酶抗酶1 功能基因 重叠延伸PCR 原核表达 |
Cloning and Prokaryotic Expression of Functional Gene of Mouse Ornithine Decarboxylase Antizyme-1 |
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| LIU Meng-yao,HAN Yu,CAI Fu-qiang,et al.Cloning and Prokaryotic Expression of Functional Gene of Mouse Ornithine Decarboxylase Antizyme-1[J].Chinese Journal of Biologicals,2010,23(2). |
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| Authors: | LIU Meng-yao HAN Yu CAI Fu-qiang |
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| Affiliation: | LIU Meng-yao,HAN Yu,CAI Fu-qiang,et al(Institute of Molecular Biology,Medical College,Three Gorges University,Yichang 443002,Hubei Province,China) |
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| Abstract: | Objective To clone the functional gene of mouse ornithine decarboxylase antizyme-1(OAZ1), express in prokaryotic cells and purify the recombinant OAZ1 protein. Methods OAZ1 gene was amplified from the total RNA of mouse melanoma B16-F1 cells by RT-PCR, and a functional gene of which the full-length could be translated without frameshifting was obtained by SOE PCR and used for construction of recombinant plasmid pET15b / OAZ1-T which was transformed to E. coli BL21(DE3)for expression under induction of IPTG.... |
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| Keywords: | Ornithine decarboxylase antizyme-1 (OAZ1) Functional gene SOE PCR Prokaryotic expression |
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