携带Foxp3基因慢病毒载体的构建

Construction of lentiviral vector carrying Foxp3 gene

目的构建携带叉头样转录因子p3(Foxp3)基因的重组慢病毒载体PWPXL-MOD-Foxp3,并包装成慢病毒,为体外获得CD4+CD2+5调节性T细胞(CD4+CD2+5 Tregs)奠定基础。方法采用双酶切法构建慢病毒表达载体PWPXL-MOD-Foxp3,用磷酸钙沉淀法将包装慢病毒所需的四质粒系统共转染人胚肾细胞293T,慢病毒系统转染48h后收集病毒上清液,纯化、浓缩后采用流式细胞术测定慢病毒滴度。结果重组的慢病毒载体滴度为3.3×108IU/ml。结论成功构建了含有Foxp3基因的高滴度的慢病毒载体,为体外获得CD 4+CD 2+5 Tregs奠定基础。

Objective To construct lentiviral vector carrying Foxp3 gene and packaged in lentivrial particles（PWPXL-MOD-Foxp3）,making the basis for obtaining CD＋4CD＋25 regulatory T cells in vitro.Methods The PWPXL-MOD-Foxp3 expression vector was be constructed by double digests.The recombinant lentiviral vector and else four packaging plasmids were co-transfected into human embroic kidney 293T cells by using calcium phosuhate technique.The supernatant was collected,purified,concentrated,and then,the viral titers were tested by flow cytometer.Result The titer of the lentiviral particles was 3.3×108 IU/ml.Conclusions The high-titer lentvirus vector containing Foxp3 geng is constructed successfully,which will contribute to obtain CD＋4CD＋25 regulatory T cells in vitro.