| 1pcDNA3.1-Ercc6l真核表达载体的构建及在P19细胞内的表达 |
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| 引用本文： | 张庆,陈祥贵.1pcDNA3.1-Ercc6l真核表达载体的构建及在P19细胞内的表达[J].泰山医学院学报,2008,29(3):164-168. |
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| 作者姓名： | 张庆 陈祥贵 |
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| 作者单位： | 1. 泰山医学院生物科学系,山东,泰安,271016
2. 西华大学生物工程学院,四川,成都,6180039 |
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| 摘 要： | 目的构建Ercc6l的正、反义真核表达载体,并在P19胚胎癌细胞内瞬时表达,在细胞学水平对基因功能进行初步研究。方法KpnⅠ/XhoⅠ双酶切pGEM-T-Ercc6l获得目的基因Ercc6l,将其克隆到同样经过双酶切的真核表达载体pcDNA3.1(+)和pcDNA3.1(-)Myc His6,重组质粒pcDNA3.1(+/-)-Ercc6l经菌落PCR和双酶切鉴定;脂质体介导法体外瞬时转染正义载体至P19细胞,RT-PCR检测基因在细胞内的表达活性情况。结果重组质粒经菌落PCR和KpnⅠ/XhoⅠ双酶切鉴定,表明真核表达载体构建正确;脂质体介导法瞬时转染正义载体至P19细胞后,检测表明转染细胞能够表达Ercc6l。结论成功构建了Ercc6l正、反义真核表达载体,其正义载体转染真核细胞后能在其中表达。
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| 关 键 词： | Ercc6l 真核表达 P19胚胎癌细胞 RT-PCR |
| 文章编号： | 1004-7115(2008)03-0164-05 |
| 修稿时间： | 2007年9月25日 |
Construction of eukaryotic expression vectors pcDNA3.1-Ercc6l and its expression in P19 cells |
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| ZHANG Qing,CHEN Xiang-gui.Construction of eukaryotic expression vectors pcDNA3.1-Ercc6l and its expression in P19 cells[J].Journal of Taishan Medical College,2008,29(3):164-168. |
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| Authors: | ZHANG Qing CHEN Xiang-gui |
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| Affiliation: | 1.Dept.of Biological Science; Taishan Medical College; Taian 271016; China; 2.Dept.of Biotechnology; Xihua University; Chengdu 610039; China); |
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| Abstract: | Objective: To construct sense and antisense eukaryotic expression vectors of novel gene Ercc6l and study its transfection and transient expression in P19 cells.Methods: The ORF of Ercc6l was obtained by digestion with XhoⅠand KpnⅠrestriction endonuclease in the plasmid of pGEM-T-Ercc6l.Recombinant vectors were constructed by inserting the target code gene into the MCS of plasmid pcDNA3.1(+) and pcDNA3.1(-) Myc His6 between XhoⅠand KpnⅠsites.Then the recombinant plasmids were identified by colony PCR and res... |
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| Keywords: | Ercc6l Eukaryotic expression P19 embryonal carcinoma cells RT-PCR |
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