小鼠甲胎蛋白基因的克隆表达鉴定及稳定表达甲胎蛋白的小鼠肿瘤细胞株的建立
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田 耕, 易继林, 刘积良, 翁 准
甲胎蛋白;小鼠;真核表达载体;细胞株,田耕,刘积良,翁准,易继林,通讯作者:,Genecloningandexpressio
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田耕, 刘积良, 翁准, 深圳市第二人民医院肿瘤科 广东省深圳市 518035
易继林, 华中科技大学同济医学院附属同济医院普外科 湖北省武汉市 430030
通讯作者: 田耕, 518035, 广东省深圳市福田区笋岗西路3002号, 深圳市第二人民医院肿瘤科. tiangeng666@yahoo.com.cn
电话: 0755-83366388-2118
收稿日期: 2005-10-14 接受日期: 2006-01-11
Gene cloning and expression identification of murinea-fetoprotein gene and establishment of a cell line stably expressinga-fetoprotein
Geng Tian, Ji-Lin Yi, Ji-Liang Liu, Zhun Weng
Geng Tian, Ji-Liang Liu, Zhun Weng, Tumor Department, Shenzhen Second People’s Hospital, Shenzhen 518035, Guangdong Province, China
Ji-Lin Yi, Department of General Surgery, Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China
Correspondence to: Dr. Geng Tian, Tumor Department, Shenzhen Second People’s Hospital, 3002 Sungang West Road, Futian District, Shenzhen 518035, Guangdong Province, China. tiangeng666@yahoo.com.cn
Received: 2005-10-14 Accepted: 2006-01-11
Abstract
AIM: To clone the murine a-fetoprotein (mAFP) gene, construct the eukaryotic expression vector of AFP and establish a cell line stably expressing AFP.
METHODS: The total RNA was extracted from Hepa1-6 cells. The mAFP gene was amplified by reverse transcription polymerase chain reaction (RT-PCR) and cloned into the eukaryotic expression vector pcDNA3.1 to construct pmAFP. The pmAFP was identified by restriction enzyme analysis and sequencing, and then stably transfected into EL-4 cell line. Western blot and RT-PCR were used to detect the expression of mAFP protein and mRNA, respectively. EL-4 cells stably expressing mAFP were inoculated in the back of 6 mice to observe the tumor formation.
RESULTS: The mAFP gene with a length of 1.8 kb was successfully cloned from the total RNA of Hepa1-6 cells. Restriction enzyme analysis and sequencing showed that the 1.8 kb mAFP gene was successfully cloned and inserted into pcDNA3 ......
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