小鼠甲胎蛋白与结核杆菌热休克蛋白70基因融合载体的构建及体外表达

Construction and expression of chimeric AFP-HSP70 plasmid in COS-7 cells

目的 构建小鼠甲胎蛋白 (α Fetoprotein ,AFP)与结核杆菌热休克蛋白 70 (Mycobacteriumtuberculosisheatshockprotein 70 ,Mt.HSP70 )基因融合载体 ,并研究其在真核细胞中的表达情况。方法 以pcDNA3.1为基本单位构建AFP及与HSP70的融合表达载体 ,融合基因以G S G G S连接子连接 ;用脂质体将系列载体导入COS 7细胞 ,4 8h后以免疫化学方法检测其表达。结果 构建的AFP和HSP70的单独及融合表达载体导入COS 7细胞 4 8h后经RT PCR检测可扩增出相应片段 ,细胞免疫化学染色为阳性。结论 AFP和HSP70的单独及融合表达载体构建成功 ,并能在真核细胞中表达。

Objective To construct chimeric vector of AFP HSP70 and investigate transientexpression of vectors in eukaryotic cells.Methods Vectors were constructed by routine molecular technique. Fusion genes were linked with G S G G S linker; Vectors were transfected into COS 7 cells by Lipofectamine 2000.MRNAs were detected by RT PCR. Proteins were assayed by immunocytochemistry.Results Proteins expressed in COS 7 cells were confirmed by RT PCR and immunocytochemistry 48 hours later.Conclusion AFP HSP70 chimeric vectors are constructed successfully and can express in eukaryotic cells.