编码hGM-CSF基因的慢病毒包装及表达

Package and expression of lentivirus encoding human GM-CSF genes

目的:构建编码人粒细胞巨噬细胞集落刺激因子(hGM-CSF)慢病毒载体转染小鼠结肠癌细胞CT26,建立持续高表达hGM-CSF基因的CT26细胞株。方法:采用PCR法获得hGM-CSF DNA片段,插入转移载体L166;用CaCl2法将载体L166-hGM-CSF、L205和L311共同转染到慢病毒包装细胞293T中,72 h后收集上清液。测定慢病毒的滴度;用ELISA法检测慢病毒感染后的CT26细胞上清液中hGM-CSF的表达。结果:通过酶切电泳证实重组转移载体L166-hGM-CSF含有预期的hGM-CSF片段。收集慢病毒转染CT26细胞,滴度达4.69×105IU/ml。ELISA法检测慢病毒转染的CT26细胞上清液中有hGM-CSF的表达超过2个月。终浓度15μg/ml的嘌呤霉素成功筛选出单克隆CT26细胞株。结论:收集的慢病毒能够有效的将其所携带的目的基因导入CT26细胞并使其在细胞中高效持续表达2个月以上。

Objective： To construct a lentiviral vector encoding human granulocyte-macrophage colony- stimulating factor（hGM-CSF） genes and to transfect CT26 cells in order to establish the CT26 strains which has been continuously over-expressing the hGM-CSF gene. Methods： DNA fragments of human GM-CSF were amplified by PCR and were inserted into the vector plasmid L166. The three plasmids expressing lentivirus were packaged into virus packaging cell line 293T using the CaC12 method. After 72 hours of transfection, the virus-producing cell supematant was produced. CT26 cells were transfected by lentivirus and the titration was determined. Results： The sequence of the PCR product in the recombinant plasmid was produced. The three plasmids were effectively transferred into 293T. When the CT26 cells were infected as target cells by the lentivirus supematant, the titration of lentivirus was 4.69× 10^5IU/ml, hGM-CSF was determined in the supematant of CT26 cells transfected by lentivirus, which was kept for more than two months. The transfected CT26 cells were screened by puromycin at a terminal concentration of 15 μg/ml. Conclusion： The lentivirus encoding hGM-CSF genes were successfully constructed.