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Position: Home > Articles > The Establishment of a Cell Line Stably Expressing Porcine GM-CSF Chinese Journal of Animal and Veterinary Sciences 2017,48 (5) 963-970

猪GM-CSF稳定表达细胞系的建立

作  者：
李俊敏;雍艳红;巩栋梁;冯园;韦璇;何玉林;巨向红
单  位：
广东海洋大学动物医学系;广东海洋大学农学院动物科学系;桂林医学院
关键词：
猪;GM-CSF;真核表达;稳定细胞系
摘  要：
旨在建立能稳定表达猪GM-CSF的细胞系。本研究利用NCBI已公布猪GM-CSF基因序列设计引物,采用RT-PCR法扩增出猪GM-CSF开放阅读框(ORF)。发现其长435bp,编码144个核苷酸的蛋白。该蛋白分子质量为16.3ku,等电点为7.15。与绵羊、马、猫、牛、狗和人的同源性分别为77.1%、73.6%、72.2%、71.5%、71.3%和70.8%。通过EcoR I和BamH I酶切后,将GM-CSF基因插入pIRES2-EGFP载体中,构建出pIRES2-EGFP-pGM-CSF重组质粒。该重组质粒转染猪肠上皮细胞(IPEC-J2)后,G418筛选阳性细胞。在转染后24h,荧光定量PCR可见GM-CSF mRNA表达量较对照组及空载体组细胞显著升高(P<0.01),荧光显微镜下亦可见大量绿色荧光表达。该细胞系在含有G418的培养液中连续传代30次,仍可见GM-CSF基因和蛋白水平的高表达。本研究成功构建了真核表达载体pIRES2-EGFP-pGM-CSF和能稳定表达猪GM-CSF的细胞系。
译  名：
The Establishment of a Cell Line Stably Expressing Porcine GM-CSF
作  者：
LI Jun-min;YONG Yan-hong;GONG Dong-liang;FENG Yuan;WEI Xuan;HE Yu-lin;JU Xiang-hong;Guilin Medical University;Department of Veterinary Medicine,Guangdong Ocean University of Agriculture;Department of Animal Science,Guangdong Ocean University of Agriculture;
关键词：
pig;;GM-CSF;;eukaryotic expression;;stable cell line
摘  要：
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 16.3ku,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 familiaris(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 hof transfection(P<0.01),and transfected cells also showed high expression of green fluorescent.After G418 selection and expansion(positiveclone)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.

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