猪GM-CSF稳定表达细胞系的建立

发布时间：2018-12-10 23:33

【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(P0.01),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]:The aim of this study was to establish a cell line capable of stably expressing porcine GM-CSF. In this study, using NCBI published porcine GM-CSF gene sequence design primers, using RT-PCR method to amplify porcine GM-CSF open reading frame (ORF). The protein encoding 144 nucleotides was found to be 435 BP long. The molecular weight of the protein is 16.3ku.The isoelectric point is 7.15. The homology with sheep, horse, cat, cattle, dog and man was 77.1% and 73.6%, 71.53% and 70.8%, respectively. After digested with EcoR I and BamH I, the GM-CSF gene was inserted into pIRES2-EGFP vector to construct pIRES2-EGFP-pGM-CSF recombinant plasmid. After the recombinant plasmid was transfected into porcine intestinal epithelial cells (IPEC-J2), positive cells were screened by G418. At 24 h after transfection, the expression of GM-CSF mRNA was significantly higher in PCR than that in control group and empty vector group (P0.01), and a large amount of green fluorescent expression was also observed under fluorescence microscope. The cell line was subcultured for 30 times in the medium containing G418, and the high expression of GM-CSF gene and protein was still observed. In this study, the eukaryotic expression vector pIRES2-EGFP-pGM-CSF and the cell line which can stably express porcine GM-CSF were successfully constructed.
【作者单位】：桂林医学院;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;
【基金】：国家自然科学基金(31101862;31472243)
【分类号】：Q78;S828

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