【摘要】 目的探讨外源性IL-2/G1融合基因在非洲绿猴肾细胞(Vero)获得稳定表达的可行性,为汉坦病毒基因工程疫苗的研制提供一定的实验基础。方法将汉坦病毒H8205株G1的cDNA重组人源IL-2基因后克隆到真核表达载体pcDNA3.1/His,形成重组真核表达质粒pcDNA3.1/His-IL-2-G1,在脂质体介导下,将其导入Vero细胞,通过G418筛选获得阳性克隆,并继续培养6周,然后通过间接免疫荧光、ELSIA、SDS-PAGE电泳等方法检测IL-2-G1基因在Vero细胞中的稳定表达情况。结果成功构建重组质粒pcDNA3.1/His-IL-2-G1,其片段插入方向正确。经间接免疫荧光和SDS-PAGE电泳证实,转染了真核表达质粒pcDNA3.1/His-IL-2-G1后在Vero细胞培养上清和胞浆中有融合蛋白的表达,其相对分子量为78000u,与预期的相符合。经人IL-2ELISA检测试剂盒检测证实在培养上清和细胞裂解物所表达的融合蛋白有IL-2特性。结论在脂质体介导下,外源性IL-2/G1融合基因能够成功导入Vero细胞并获得稳定表达。更多还原

【Abstract】 Objective To explore the feasibility of stable expression of Hantavirus G1and huma n IL-2fusion gene in Vero cells.Methods pcDNA3.1/His-IL-2-G1was constructed.With the help of lipofectamine,the cultured Vero cells were transfected with pcDNA3.1/His-IL-2-G1and the positive cells were selected by G418.IFAT,ELISA and SDS-PAGE electrophoresis were used to determ ine the stable transfection and expr ession of recombinant protein.Results The positive recombinant plasmid harbouring the right inserted G1/IL-2fusion gene was obtained.The fusion pro tein expressed in Vero cells was 78000u,corresponding to the estimated molecular size,and the IL-2domain was confirmed by ELISA.Conclusion With the help of lipofectamine,IL-2/G1fusion gene could be transferred and stably expressed the IL-2-G1fusion protein in Vero cells.更多还原