α-HNP-1转基因细胞系的建立  

Establishment of Humanα-HNP-1 Stable Transfected Epithelial Cell Line Derived form Mouse Embryonic Stem Cells

作　　者：高其双 黄海军[2] 欧阳静萍[3] 吴建英 陶弼菲 杨悦[3] 张德玲[3] 蒋思文[2] 

机构地区：[1]武汉市畜牧兽医科学研究所,武汉430065 [2]华中农业大学农业动物遗传育种与繁殖教育部重点实验室&农业部猪遗传育种重点开放实验室,武汉430070 [3]武汉大学医学院过敏及免疫相关疾病重点实验室,武汉430071

出　　处：《生物技术通报》2007年第6期113-116,共4页Biotechnology Bulletin

基　　金：湖北省科技攻关计划课题(2003AA303B06)

摘　　要：目的构建稳定表达人α-HNP-1的转基因细胞系,为稳定生产α-HNP-1并将其应用于医药开发提供生产细胞源。方法真核表达载体pcDNA3.1(-)/HNP-1经酶切和测序鉴定后,用脂质体转染法转染昆明白小鼠胚胎干细胞来源的上皮细胞,通过不同浓度的G418加压筛选,建立稳定转染的胚胎干细胞来源的上皮细胞系,用RT-PCR及抑菌试验检测α-HNP-1的表达。结果建立了稳定转染的ES来源的上皮细胞系,成功地表达目的基因,其培养上清液及细胞冻融液具有抑菌作用,结论真核表达载体稳定转染胚胎干细胞来源的上皮细胞系,为进一步研究α-HNP-1的功能奠定了基础。Objective To transfect embryonic stem cells derived epithelium cells with eukaryotic expression vector containing goat beta-lactoglobulin （BLG）gene promoter and human α-defensin-1 （α-HNP-1）gene to establish stable transfected epithelial cell line, Methods After the identification by digestion and sequencing on the recombinant α karyotic expression vector pcDNA3.1 （＋）/BLG-HNP-I,the recombinant was transfected into embryonic stem cells derived epithelial cells by lipofectamineTM 2000. After screening culture by G418,stable transfected epithelial cell line was established,and the transcription and expression of α-HNP-1 were identified by RT-PCR. The antibacterial activities of cellular soluble protein and culture supernatant were examined in vitro. Results The stable transfected embryonic stem cells derived epithelial cell line was established. The α-HNP-1 protein was expressed successfully. Conclusion The establishment of stable embryonic stem cells derived epithelial cell line provide solid foundation for further experimental studies on the function of ot-HNP-1.

关 键 词：人α-防御素-1 真核表达载体 ES细胞 上皮细胞系 稳定转染 基因表达 

分 类 号：Q78[生物学—分子生物学]