稳定转染pGM-CSF基因的哺乳动物细胞系的建立  

Establishment of mammalian animal cell line stably transfected with porcine GM-CSF gene

作　　者：陶弼菲 高其双 陈志华 向敏 占才耀 钱运国 夏瑜 华娟 王莲芳 任远 黄海军 

机构地区：[1]武汉市畜牧兽医科学研究所,湖北武汉430208

出　　处：《中国兽医杂志》2013年第8期6-8,12,共4页Chinese Journal of Veterinary Medicine

基　　金：国家自然科学基金项目(31201938);武汉市晨光计划项目(201150431078);武汉市农科院青年英才计划项目(YC201101)

摘　　要：构建稳定表达猪pGM-CSF基因的细胞系可以为工业化生产猪pGM-CSF并将其应用于新型兽药的开发提供生产细胞源。本试验用脂质体介导法将真核表达载体plRES2-EGFP-pGM-CSF瞬时转染BHK-21细胞,通过不同浓度的G418加压筛选和进一步采用单个克隆法筛选,建立稳定转染且高效表达pGM-CSF的BHK-21细胞株9个,采用Elisa方法测定稳定转染pGM-CSF的BHK-21细胞株细胞培养上清液中pGM-CSF的表达水平达到329.37±13.76ng/mL。为下一步从细胞培养物中大量提取目的蛋白并最终开发出一种用于提高动物疫苗免疫效力的新型生物制剂奠定基础。Establishment of a stable cell line expression pig pGM-CSF can provide cell source for indus- trial produce of pig pGM-CSF and is applied to the development of new veterinary drug production. After identification by digestion and sequencing on the recombinant eukaryotic expression vector pIRES2-EGFP- pGM-CSF, the recombinant was transfected into BHK-21 cell by Lipofectamine 2000. After screening cul- ture by G418, nine stable transfected cell strain was established, and the transcription and expression of pGM-CSF were identified by RT-PCR. The pGM-CSF protein in culture supernatant were examined by ELISA, and the concentration is 329.37＋ 13.76 ng/mL. The establishment of stable BHK-21 cell line provides solid foundation for further experimental studies on the biological function of pGM-CSF and the application in veterinary biological products.

关 键 词：猪粒细胞-巨噬细胞集落刺激因子 哺乳动物细胞 稳定转染 蛋白表达 

分 类 号：S858.28[农业科学—临床兽医学]