猪GM-CSF稳定表达细胞系的建立

doi:10.11843/j.issn.0366-6964.2017.05.022

摘要/Abstract

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435 bp，编码144个核苷酸的蛋白。该蛋白分子质量为16.3 ku，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24 h，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。

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 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 GM-CSF 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.

中图分类号:

S826.2

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.

LI Jun-min, YONG Yan-hong, GONG Dong-liang, FENG Yuan, WEI Xuan, HE Yu-lin, JU Xiang-hong. The Establishment of a Cell Line Stably Expressing Porcine GM-CSF[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(5): 963-970.

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