畜牧兽医学报2017，Vol.48Issue(4)：637-644,8.DOI:10.11843/j.issn.0366-6964.2017.04.006
绵羊NYD-SP27基因在BHK-21细胞中的稳定表达及其特征鉴定
Construction and Identification of BHK-21 Cell Line Stably Expressing Ovine NYD-SP27
摘要
Abstract
In order to study function of ovine NYD-SP27,BHK-21 cell line stably expressing ovine NYD-SP27 was constructed and relative phenotypic characteristics were analyzed,which lay a foundation for function analysis of ovine NYD-SP27 gene.The ovine NYD-SP27 gene was cloned and inserted into eukaryotic expressing vector pDsRed1-C1.To co-express Red fluorescent protein gene and NYD-SP27 gene,both of them were linked with porcine teschovirus-1 2A peptide (P2A).The recombinant plasmid pDsRed-P2A-Flag-NYDSP was transfected into BHK-21 cells and selected with G418.Positive cells were identified using RT-PCR,indirect immunofluorescence assay (IFA) and Western blot.The RT-PCR results indicated that NYD-SP27 gene was stably integrated into genome of BHK-21 cells.The NYD-SP27 protein could be expressed in BHK-21 cell line and the molecular weight was approximately 63 ku,which suggested that the self-cleavage of P2A realized during the translation process of NYD-SP27 protein and Red fluorescent protein.The eukaryotic expression vectors were confirmed successfully and the BHK-21 cell lines stably expressing ovine NYD-SP27 was successfully established in this study.关键词
绵羊NYD-SP27基因/稳定表达/BHK-21细胞/P2AKey words
ovine NYDSP27 gene/stably expressing/BHK-21 cells/P2A分类
农业科技引用本文复制引用
马亚茹,胡广东,王红红,加米拉,徐锦凤,陈创夫,赛务加甫..绵羊NYD-SP27基因在BHK-21细胞中的稳定表达及其特征鉴定[J].畜牧兽医学报,2017,48(4):637-644,8.基金项目
国家自然科学基金项目(31460683) （31460683）
