小鼠Foxp3基因慢病毒载体构建及其在DC2.4细胞的表达  

Construction of lentiviral expression vector containing Foxp3 gene and its expression in DC2.4 cell line

作　　者：宫玉波[1] 黄一飞[1] 黎燕[2] 韩根成[2] 刘勇[1] 王大江[1] 杜改萍[1] 余继锋[1] 

机构地区：[1]解放军总医院眼科,北京100853 [2]军事医学科学院基础医学研究所分子免疫室,北京100850

出　　处：《细胞与分子免疫学杂志》2011年第7期721-724,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(30973245);中国博士后科学基金资助项目(20100471821)

摘　　要：目的:构建小鼠Foxp3基因的慢病毒载体,体外感染树突状细胞DC2.4,制备Foxp3+DC细胞,为进一步研究其免疫调节作用奠定基础。方法:将小鼠Foxp3基因克隆到慢病毒pGC-FU载体,通过PCR和测序鉴定获得连接正确的克隆。将鉴定后的重组表达质粒pGC-FU-Foxp3与pHelper1.0质粒和pHelper2.0质粒共转染293T细胞,制备携带Foxp3基因的慢病毒Lentivirus-Foxp3。Lentivirus-Foxp3感染体外培养的DC细胞,流式细胞术(FCM)检测感染后的DC细胞Foxp3表达。结果:构建的慢病毒载体pGC-FU-Foxp3经PCR鉴定和测序正确,包装慢病毒Lentivirus-Foxp3滴度为2×108TU/mL。慢病毒Lentivirus-Foxp3感染DC2.4细胞后Foxp3表达明显增加。结论:成功构建了小鼠Foxp3基因的慢病毒表达载体,慢病毒Lentivirus-Foxp3能有效感染DC细胞。AIM：To construct a Foxp3 lentiviral vector and transfer it into DC2.4 cells,which provides Foxp3＋DC cells for further study on its immune modulation.METHODS：We cloned mouse Foxp3 gene into lentiviral vector（pGC-FU） and acquired the plasmid pGC-FU-Foxp3.PCR and sequencing analysis were made for verifying the positive clones.The virus packaging plasmids（pGC-FU-Foxp3,pHelper1.0 and pHelper2.0） were contransfected into 293T cells,and the Lentivirus-Foxp3 was harvested from 293T cells.The Lentivirus-Foxp3 was used to infect DC2.4 cells in vitro and the expression of Foxp3 in infected DC2.4 cells was detected with flow cytometry（FCM）.RESULTS：PCR and sequencing revealed that the pGC-FU-Foxp3 plasmid was correctly constructed.The Lentivirus-Foxp3 with a titer of 2×108 TU/mL was successfully packaged.Foxp3 expression in DC2.4 cells infected with the Lentivirus-Foxp3 was increased significantly compared with negative control lentivirus.CONCLUSION：The pGC-FU-Foxp3 plasmid has been successfully constructed and the Lentivirus-Foxp3 has been successfully packaged.Foxp3 can be enhanced in DC cells infected with the Lentivirus-Foxp3.

关 键 词：FOXP3 慢病毒 DC2.4 

分 类 号：R392.12[医药卫生—免疫学]