稳定表达新城疫病毒V蛋白禽源细胞系的建立  

Establishment of stable avian cell line expressing V protein of Newcastle disease virus

作　　者：唐文雅[1] 王兴龙[1] 张渭东[1] 杜恩岐[1] 陈胜利[1] 王丹阳[1] 党如意[1] 张淑霞[1] 杨增岐[1] 

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100

出　　处：《中国兽医科学》2012年第11期1152-1157,共6页Chinese Veterinary Science

基　　金：陕西省农业科技创新项目(2011NXC01-10)

摘　　要：为获得可以稳定表达新城疫病毒V蛋白的DF-1细胞系,以质粒载体为模板,用PCR方法扩增新城疫病毒V基因并引入酶切位点(EcoRⅠ和BamHⅠ),以同尾酶相连的方式与用EcoRⅠ和BglⅡ酶切的piggyBac转座子系统克隆质粒pTKL1-CMV-Puro-EGFP相连接,经酶切鉴定、测序分析正确后,用Lipo-fectamineTM2000将重组质粒pTKL1-CMV-Puro-EGFP-V和辅助质粒mPB共转染DF-1细胞,通过绿色荧光观察、嘌呤霉素筛选及蛋白表达的Western-blot检测,结果表明,稳定表达新城疫病毒V蛋白的DF-1细胞系已成功建立。证实piggyBac转座子系统能够用于稳定表达外源基因的禽源细胞系的构建,为piggy-Bac转座子系统在禽源细胞的应用和新城疫病毒V蛋白的研究提供了一定的参考资料。In order to obtain the stable DF-1 cell lines expressing Newcastle disease virus（NDV） V protein,NDV V gene from the recombinant plasmid pEGFT-N1-V was amplified, the restriction sites of EcoR l and BamH I were inducted into the amplicon. The amplicon were then connected into the piggy- Bac transposon cloning plasmid pTKL1-CMV-Puro-EGFP vector,which was checked by enzyme digestion and sequence analysis. The recombinant plasmids were co-transfected with the helper plasmid mPB into DF-1 cells using LipofectamineTM2000. The cell line was selected with puromycin and detected with green fluorescent protein observation, and V protein expression was detected by Western-blot. Results showed that the stable monoclonal cell lines expressing V protein was constructed successfully. The results revealed that piggyBac transposon system can be used for the construction of avian stable cell lines and the DF-1 cell line（DF-1-V） expressing NDV V protein was established successfully. The present study provided reference materials for application of the piggyBac transposon system in avian cell and research of NDV V protein.

关 键 词：PIGGYBAC转座子 新城疫病毒V蛋白 稳定表达 细胞系 

分 类 号：S852.659.5[农业科学—基础兽医学]