甲胎蛋白与热休克蛋白70基因融合载体的构建及表达

Construction and expression of secreted chimericAFP-HSP70 plasmid in eukaryotic cells

目的 :构建分泌性小鼠甲胎蛋白 (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 pSecTag2 B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 ,抽提总RNA作RT -PCR测其在转录水平的表达。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后经细胞免疫化学染色为阳性 ,RT -PCR可扩增出特异性条带。结论 :AFP和HSP70的单独及融合表达载体构建成功 ,能在真核细胞中表达。

Objective:To construct secreted chimeric vector of AFP-HSP70 and investigate transient expression 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. Proteins were assayed by immunocytochemistry and RT-PCR for its RNA.Results:Proteins expressed in COS-7 were comfirmed by immunocytochemistry 48 hours later and RNA was detected by RT-PCR.Conclusion:AFP-HSP70 chimeric vector is constructed successfully and could be expressed in eukaryotic cells.