HBD_3基因乳腺特异性表达载体构建及真核表达.pdfVIP

生 物 工 程 学 报 Chin J Biotech 2009, July 25; 25(7): 968-974 Chinese Journal of Biotechnology ISSN 1000-3061 cjb@ © 2009 Institute of Microbiology, CAS CSM, All rights reserved 动物及兽医生物技术 HBD-3 彭巍, 兰志刚, 马晶晶, 王保垒, 张涌 , 712100 : 为了制备高效表达人β-防御素-3 基因的转基因奶牛提供可靠的核移植供体细胞, 本试验采用RT-PCR 从人胎盘 组织获得人β-防御素3 cDNA, 通过双酶切法将目的基因片段hBD 插入到质粒pBCP 之间, 最后再通过双酶切法将目的 片段BCD 插入到真核表达载体pEGFP-C1 中, 最终构建乳腺特异性表达载体pEBCD 。脂质体介导法转染奶牛胎儿成纤 维细胞G418, 抗性筛选 3~4 周后, 经PCR 、RT-PCR 扩增检测和报告基因 EGFP 表达检测, 得到稳定整合外源基因的 β 转基因供体细胞; 同时检测稳定转染的奶牛乳腺上皮细胞系的上清液, 检测到重组人 -防御素-3 蛋白可以在奶牛乳腺 上皮细胞组织特异性表达。 : 人β- 防御素 3, 奶牛β-酪蛋白, EGFP 报告基因, 供体细胞, 核移植 Construction of HBD-3 gene mammary-specific expression vector and eukaryotic expression Wei Peng, Zhigang Lan, Jingjing Ma, Baolei Wang, and Yong Zhang Institute of Biotechnology, College of Animal Veterinary Medicine, Northwest Agiculture and Forestry University, Yangling 712100, China Abstract: To establish human β-defensin-3 gene transgenic cell lines as competent donor cells for the production of transgenic animals using somatic cell nuclear transfer (SCNT). Firstly, we obtained human β-defensin-3 by RT-PCR from human placenta, and subsequently inserted the fragment hBD into the corresponding site of the plasmid pBCP. Then we moved the combined fragment BCD (including 5′ and 3′ regulating region of β-casein and hBD) into the corresponding site of the plasmid pEGFP-C1. Finally we successfully constructed mammary-specific expression vector pEBCD. We transected pEB