| 小鼠OAZ2基因的原核表达及抗体制备 |
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| 引用本文： | 刘梦瑶,韩钰,何玲,蔡富强,王艳林. 小鼠OAZ2基因的原核表达及抗体制备[J]. 生物技术, 2011, 21(3). DOI: 10.3969/j.issn.1004-311X.2011.03.064 |
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| 作者姓名： | 刘梦瑶 韩钰 何玲 蔡富强 王艳林 |
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| 作者单位： | 三峡大学分子生物学研究所,三峡大学医学院,湖北宜昌,443002 |
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| 基金项目： | 国家自然科学基金项目，湖北省教育厅重点研究项目 |
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| 摘 要： | 目的:克隆小鼠鸟氨酸脱羧酶抗酶2(OAZ2)功能基因,原核表达、纯化OAZ2蛋白并制备抗OAZ2多克隆抗体.方法:IRT-PCR法从鼠黑色素瘤细胞总RNA中克隆OAZ2 cDNA后,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.将OAZ2功能基因克隆人原核表达载体pET15b并原核表达.表达的蛋白经Ni-NTA亲和层析纯化后,用SDS-PAGE和Western Blot分析鉴定.用纯化的OAZ2蛋白作为抗原免疫Bab/C小鼠以制备多克隆抗体,制备抗体用ELISA和Western Blot检测抗体滴度和特异性.结果:成功获得小鼠OAZ2 cDNA并构建出无需移码翻译的OAZ2功能基因.OAZ2功能基因在大肠杆菌BL21(DE3)中可诱导性高表达并能用Ni-NTA树脂高效纯化.用纯化蛋白免疫Bab/C小鼠制备的抗血清经ELISA检测有较高的多克隆抗体效价(＞1∶64000),经Western blot鉴定可与纯化的OAZ2蛋白质特异性结合.结论:建立了鼠OAZ2蛋白原核表达和纯化技术,制备出高效价和特异性抗OAZ2多克隆抗体,为进一步研究OAZ2基因的功能奠定了基础.
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| 关 键 词： | 鸟氨酸脱羧酶抗酶 重叠延伸PCR 原核表达 抗体 |
Prokaryotic Expression of Mouse OAZ2 and Preparation of Its Antibody |
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| LIU Meng-yao,HAN Jue,HE Ling,CAI Fu-qiang,WANG Yang-lin. Prokaryotic Expression of Mouse OAZ2 and Preparation of Its Antibody[J]. Biotechnology, 2011, 21(3). DOI: 10.3969/j.issn.1004-311X.2011.03.064 |
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| Authors: | LIU Meng-yao HAN Jue HE Ling CAI Fu-qiang WANG Yang-lin |
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| Affiliation: | LIU Meng-yao,HAN Jue,HE Ling,CAI Fu-qiang,WANG Yang-lin*(Institute of Molecular Biolgy,Medical College,China Three Gorges University,Yichang 443002,China) |
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| Abstract: | Objective: To clone functional gene of mouse ornithine decarboxylase antizyme-2(OAZ2),to express and purify recombinant OAZ2 in prokaryotic system,and then to generate anti-OAZ2 polyclone antibody.Method: OAZ2 cDNA was amplified by RT-PCR from total RNA of mouse melanoma B16-F1 cells.The function gene that can translate whole length OAZ2 without frameshifting was obtained by PCR-driven overlap extension.The function gene was then subcloned into the plasmid pET15b and was transformed into Bl21(DE3) host cell... |
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| Keywords: | OAZ2 PCR-driven overlap extension prokaryotic expression antibody |
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