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| 可调控的鼠白细胞介素-12双亚基共表达质粒的构建及在体外表达 | |
| 引用本文： | 陈坚,张斌,薛绪潮,方国恩,苏长青,钱其军. 可调控的鼠白细胞介素-12双亚基共表达质粒的构建及在体外表达[J]. 中国普外基础与临床杂志, 2008, 15(12) |
| 作者姓名： | 陈坚 张斌 薛绪潮 方国恩 苏长青 钱其军 |
| 作者单位： | 1. 解放军第81医院肿瘤外科,南京,210002 2. 第二军医大学附属长海医院普通外科,上海,200433 3. 第二军医大学附属东方肝胆外科医院病毒基因治疗实验室,上海,200438 |
| 基金项目： | 国家自然科学基金资助项目 |
| 摘 要： | 目的构建可经米非司酮（RU486）调控表达小鼠白细胞介素-12（mIL-12）单链融合基因的真核载体，并鉴定其调控表达效果。方法以GCp35Ep40PN质粒为模板，分别获取mIL-12 p40和p35亚基的基因，重叠PCR引入linker后，克隆人pCA14质粒，测序正确之后，装人可调控载体pRS-17而构建成真核表达质粒pRS-RUmIL-12。用Lipofectamine 2000将pRS-RUmIL-12质粒转染HEK293细胞，以不同剂量的RU486诱导其表达，然后用ELISA法检测其培养上清液中mIL-12蛋白的含量。结果所得mIL-12单链融合基因序列与设计一致。pRS-RumIL-12在体外转染HEK293细胞后，ELISA检测结果表明该系统具有良好的调控能力：无诱导剂RU486时，mIL-12蛋白表达量很低，而加入诱导剂RU486后，可以诱导mIL-12的表达，并在一定范围内mIL-12的蛋白表达量与诱导剂的浓度呈正相关。结论成功构建了可经RU486调控的mIL-12双亚基共表达的真核表达质粒，可用于进一步的基因调控和基因治疗研究。 |
| 关 键 词： | 低氧诱导因子-1α 巢式PCR Tet-on基因表达系统 反应质粒 克隆 |
Construction of Regulatable Murine IL-12 Eukaryotic Expression Plasmid of Single Chain Fusion Gene and Identification of Its Expression in Vitro |
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| CHEN Jian,ZHANG Bin,XUE Xu-chao,FANG Guo-en,SU Chang-qing,QIAN Qi-jun. Construction of Regulatable Murine IL-12 Eukaryotic Expression Plasmid of Single Chain Fusion Gene and Identification of Its Expression in Vitro[J]. Chinese Journal of Bases and Clinics In General Surgery, 2008, 15(12) | |
| Authors: | CHEN Jian ZHANG Bin XUE Xu-chao FANG Guo-en SU Chang-qing QIAN Qi-jun |
| Affiliation: | CHEN Jian1,ZHANG Bin1,XUE Xu-chao2,FANG Guo-en2,SU Chang-qing3,QIAN Qi-jun3. 1.Department of Oncosurgery,The 81st Hospital of PLA,Nanjing 210002,China,2.Department of General Surgery,Changhai Hospital,The Second Military Medical University,Shanghai 200433,3.Laboratory of Viral , Gene Therapy,Eastern Hepatobiliary Surgical Hospital,Shanghai 200438 |
| Abstract: | Objective To construct a regulatable plasmid containing single chain fusion gene of murine interleukin-12(mIL-12)which was regulated with mifepristone(RU486)and explore its expression in vitro.Methods The p40 and p35 subunit sequence of mIL-20 were respectively obtained from the plasmid GCp35Ep40PN by polymerase chain reaction(PCR)and they were cloned into pCA14 plasmid after introducing a linker by overlap PCR.The single chain mIL-12 gene was comfirmed by sequencing and subcloned into pRS-17 vector which c... |
| Keywords: | Interleukin-12 Fusion gene Eukaryotic expression Mifepristone |
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