猪GM-CSF稳定表达细胞系的建立

The Establishment of a Cell Line Stably Expressing Porcine GM-CSF

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。

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.