中国畜牧兽医2017，Vol.44Issue(8)：2261-2268,8.DOI:10.16431/j.cnki.1671-7236.2017.08.007
猪流行性腹泻病毒CH-HuN1301株N基因重组表达载体的构建及稳定表达细胞系的建立
Construction of Recombinant Expression Vector and Establishment of Stable Expression Cell Line of N Gene of Porcine Epidemic Diarrhea Virus Strain CH-HuN1301
摘要
Abstract
This study was aimed to construct stable cell line expressing N protein coded by porcine epidemic diarrhea virus(PEDV)strain CH-HuN1301.N gene of this virus strain was amplified by RT-PCR,which was then cloned into prokaryotic expression vector pET28a and eukaryotic expression vector pEGFP-C1,respectively.Recombinant plasmids were sequenced and analyzed by software Mega 5.0.To prepare polyclonal antibody of N protein,the prokaryotic expression recombinant N protein was used as antigen to immunize rabbits.HEK293 transfected with recombinant eukaryotic expression plasmids pEGFP-PEDV-N was selected by G418 to produce a stable cell line which then was identified by immunoperoxidase monolayer assay(IPMA)and fluorescence observation.Results showed that,the N gene of PEDV strain CH-HuN1301 was 1 323 bp,molecular weight of recombinant prokaryotic expressing N protein was about 60 ku,prepared polyclonal antibody against N protein showed wonderful reactivity with PEDV propagated in Vero cells,recombinant eukaryotic expressing N protein was positively detected in the established stable cell line by IPMA and fluorescence observation.This stable cell line provided foundation for the research in PEDV diagnosis methods.关键词
猪流行性腹泻病毒/CH-HuN1301毒株/N基因/稳定细胞系Key words
porcine epidemic diarrhea virus (PEDV)/CH-HuN1301/N gene/stable cell line分类
农业科技引用本文复制引用
唐青海,杨海,黎露,陈果亮,何丽芳,刘最,王芳宇..猪流行性腹泻病毒CH-HuN1301株N基因重组表达载体的构建及稳定表达细胞系的建立[J].中国畜牧兽医,2017,44(8):2261-2268,8.基金项目
国家自然科学基金青年基金(31101837) （31101837）
衡阳师范学院引进人才专项项目(16D20) （16D20）
湖南省教育厅优秀青年项目(17B038) （17B038）
