| 小鼠Lin28基因的克隆及原核表达 |
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| 引用本文： | 宁方勇,王春生,张秋婷,安星兰,朴善花,安铁洙. 小鼠Lin28基因的克隆及原核表达[J]. 中国实验动物学报, 2011, 19(6): 456-460. DOI: 10.3969/j.issn.1005-4847.2011.06.002 |
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| 作者姓名： | 宁方勇 王春生 张秋婷 安星兰 朴善花 安铁洙 |
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| 作者单位： | 1. 东北林业大学生命科学学院,哈尔滨 150040;东北农业大学动物科技学院,哈尔滨 150030
2. 东北林业大学生命科学学院,哈尔滨,150040 |
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| 基金项目： | 国家自然科学基金资助项目，中央高校基本科研业务费专项资金资助 |
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| 摘 要： | 目的探讨获取小鼠Lin28蛋白的方法。方法 提取8.5 d ICR小鼠胚胎mRNA后反转录为cDNA序列,用一对两端引入特定酶切位点（NcoⅠ及XhoⅠ）引物,从该cDNA中扩增出Lin28基因编码区序列;将获得的Lin28基因编码区序列克隆到pMD18-T载体上。对质粒双酶切回收其中Lin28基因片段,与pET-30a（+）载体相连接并转化Rosetta（DE3）型大肠杆菌,用IPTG诱导表达,最后采用SDS-PAGE对表达结果进行分析。结果对所克隆的Lin28蛋白编码区的DNA序列分析表明,Lin28 CDS区包括终止密码子在内为630 bp,与参照DNA（NM145833）相比同源性为99.37%,与参照氨基酸序列相比同源性为100%;在IPTG诱导下pET-30a（+）-Lin28重组质粒可表达与预期相符的约为27.5×103的蛋白质。结论利用克隆的小鼠Lin28基因,采用原核表达方法,成功获得小鼠Lin28蛋白,为进一步开展以重组蛋白诱导体细胞重编程研究奠定基础。
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| 关 键 词： | ICR小鼠 Lin28 克隆 原核表达 |
Cloning and prokaryotic expression of mouse Lin28 gene |
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| NING Fang-yong,WANG Chun-sheng,ZHANG Qiu-ting,AN Xing-lar,PIAO Shan-hua,AN Tie-zhu. Cloning and prokaryotic expression of mouse Lin28 gene[J]. Acta Laboratorium Animalis Scientia Sinica, 2011, 19(6): 456-460. DOI: 10.3969/j.issn.1005-4847.2011.06.002 |
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| Authors: | NING Fang-yong WANG Chun-sheng ZHANG Qiu-ting AN Xing-lar PIAO Shan-hua AN Tie-zhu |
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| Affiliation: | NING Fang-yong1,2,WANG Chun-sheng1,ZHANG Qiu-ting1,AN Xing-lan1,PIAO Shan-hua1,AN Tie-zhu1(1.College of Life Science,Northeast Forestry University,Harbin 150040,China,2.College of Animal Science and Technology,Northeast Agricultural University,Harbin 150030) |
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| Abstract: | Objective Lin-28 is a gene recently shown to be involved in the conversion of somatic cells to induced pluripotent stem cells.The aim of this study was to clone Lin28 gene and analyzed its expression in E.coli so to make an effort on the further study of its function.Methods Lin28 gene was cloned from the cDNA amplified from 8.5-day mouse embryos mRNA,and ligated with linear vector pMD18-T.The recombinant plasmid was digested and the segment with Lin28 gene was purified then subcloned into prokaryotic expre... |
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| Keywords: | ICR mouse Lin28 gene Clone Prokaryotic expression |
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