中国兽医科学2017，Vol.47Issue(4)：419-426,8.DOI:10.16656/j.issn.1673-4696.2017.04.003
慢病毒载体介导稳定表达pAPN的Vero细胞系的构建及应用
Establishment and application of a Vero cell line with stably expressing porcine APN gene by lentiviral vector
摘要
Abstract
The porcine aminopeptidase N sequence was cloned into the lentiviral vector pLVX-mCherry via homologous recombination.The recombinant plasmid pLVX-mCherry-pAPN was transfected into 293T with lentiviral packaging plasmids PLP1,plp2,PLP/VSVG,respectively.The recombinant virus was harvested and Vero cells were infected.The cells expressing pAPN were selected by culturing in nutrient solutions containing puromycin and limitied dilution.The expression of APN in the cell line was identified by RT-PCR and IFA.A monoclonal cell line was successfully obtained using single cell cloning technology and,was named Vero-APN-B6.The susceptibility to porcine epidemic diarrhea virus(PEDV) of this cell line,compared with the normal Vero cells,was detected by the methods of IFAand determination of TCID50.The results showed that the cell line was more susceptible to PEDV than the normal Vero cells.PEDV SC-MS stain was isolated from small intestinal tissues using this cell line.关键词
pAPN/细胞系/慢病毒载体/PEDV/SC-MS株Key words
porcine aminopeptidase N/cell line/lentiviral vector/porcine epidemic diarrhea virus/SC-MS stain分类
农业科技引用本文复制引用
陆倩倩,王广操,许长萌,王志建,王先炜..慢病毒载体介导稳定表达pAPN的Vero细胞系的构建及应用[J].中国兽医科学,2017,47(4):419-426,8.基金项目
南京农业大学科研项目(KJQN201619) （KJQN201619）
