畜牧兽医学报2017，Vol.48Issue(5)：963-970,8.DOI:10.11843/j.issn.0366-6964.2017.05.022
猪GM-CSF稳定表达细胞系的建立
The Establishment of a Cell Line Stably Expressing Porcine GM-CSF
摘要
Abstract
This study was conducted to establish cells lines stably expressing porcine GM-CSF.The primer of porcine GM-CSF was designed according to the sequence published in NCBI,the open reading frame (ORF) was cloned by RT-PCR method.The porcine GM-CSF gene cloned consisted of 435 bp,and the ORF of porcine GM-CSF encoded a protein of 144 amino acids.The GM-CSF polypeptide had an estimated molecular mass of 16.3 ku,PI of 7.15 and displayed a high homology to Ovis aries(77.1％),Equus caballus (73.6％),Felis catus(72.2％),Bos taurus(71.5％),Canis lupus f amiliaris (71.3 ％)and Homo sapiens (70.8％),respectively.After the digestion of pIRES2-EGFP vector by EcoR I and BamH I enzyme,the ORF of porcine GMCSF was inserted and named pIRES2-EGFP-pGM-CSF.Following,the pIRES2-EGFP-pGM-CSF was transfected into porcine intestinal epithelial cell line IPEC-J2,which was then screened by G418.Consequently,the expression of GM-CSF mRNA in transfected cells was significantly increased compared to control cells and after 24 h of transfection(P＜0.01),and transfected cells also showed high expression of green fluorescent.After G418 selection and expansion (positive clone) for 30 passages,the expression of GM-CSF mRNA was increased to control compared cells.We concluded that the eukaryotic expression vector pIRES2-EGFP-pGM-CSF was constructed successfully and a cell lines that show the stable expression of porcine GM-CSF was established.关键词
猪/GM-CSF/真核表达/稳定细胞系Key words
pig/GM-CSF/eukaryotic expression/stable cell line分类
农业科技引用本文复制引用
李俊敏,雍艳红,巩栋梁,冯园,韦璇,何玉林,巨向红..猪GM-CSF稳定表达细胞系的建立[J].畜牧兽医学报,2017,48(5):963-970,8.基金项目
国家自然科学基金(31101862 （）
31472243) （）
