表达ESAT-6-gpi和IL-21的B16F10瘤苗的构建及其活性鉴定  

Construction of B16F10 tumor vaccine expressing ESAT-6-gpi and IL-21 and identification of its biological activity

作　　者：何向锋[1] 王净[2] 余方流[1] 赵枫姝[1] 张洪义[1] 曹文虎[1] 窦骏[1] 

机构地区：[1]东南大学医学院病原生物学与免疫学系 [2]东南大学附属中大医院妇产科,南京210009

出　　处：《现代免疫学》2011年第5期358-362,共5页Current Immunology

基　　金：江苏省六大人才高峰项目(医药行业D14);国家自然科学基金资助项目(81071769)

摘　　要：为构建膜表达糖基化磷脂酰肌醇(GPI)锚定的结核杆菌早期分泌靶抗原6 kD(ESAT-6)和分泌IL-21的B16F10瘤苗并鉴定其活性,利用重叠PCR法构建pIRES-ESAT-6-gpi/IL-21重组质粒,以脂质体转染重组质粒到B16F10细胞,G418筛选出阳性克隆,用RT-PCR、免疫荧光、FCM和Western blot检测瘤苗细胞靶抗原表达,用瘤苗细胞培养上清刺激小鼠CD8+T细胞,检测瘤苗所分泌IL-21的生物学活性。结果表明,pIRES-ESAT-6-gpi/IL-21重组质粒DNA测序正确,B16F10-ESAT-6-gpi/IL-21瘤苗细胞目的基因ESAT-6表达于瘤苗细胞表面,增殖能力未受外源基因导入影响,分泌的IL-21具有生物学活性,为研究膜表达ESAT-6和分泌表达IL-21瘤苗的抗瘤效应奠定了基础。To construct B16F10 tumor vaccine that express the IL-21 and the 6 kD early secreted antigenic target（ESAT-6） anchored by glycosylated phosphatidylinositol（GPI） on cell membrane and to identify its biological activity,plasmid recombination method based on overlap extension PCR was used to construct the eukaryotic expression vector for pIRES-ESAT-6-gpi/IL-21,then the melanoma B16F10 cells were transfected with the recombinant plasmid.The expressions of the fused protein and IL-21 were determined by RT-PCR,immunofluorescence,FCM,Western blot assay,respectively.Mouse splenocytes were cultivated with supernatant from the tumor vaccine cells to detect the biological activity of IL-21 secreted by tumor vaccine cells.It was demonstrated that the ESAT-6 target molecule was expressed on the surface of B16F10-ESAT-6-gpi/IL-21 tumor vaccine cells,and the exogenous gene did not affect the proliferation of vaccine cells.Furthermore,the IL-21 secreted from vaccine cells has biological activity.The stable transfected B16F10-ESAT-6-gpi/IL-21 tumor vaccine cell line was successfully constructed and the vaccine could be used for further anti-tumor research.

关 键 词：B16F10 IL-21 ESAT-6 GPI 肿瘤疫苗 

分 类 号：R392[医药卫生—免疫学]