【摘要】 目的为探讨GJB6的组装、运输、膜定位及间隙连接通道形成的可能机制,在hela细胞筛选GJB6的C-端功能域的互作蛋白。方法以正常人DNA为模板,PCR扩增GJB6(CX30)的211~261氨基酸编码区,即C-端功能域,构建pGEX-4T-2-GJB6(211~261)-GST原核融合表达载体,纯化融合蛋白和GST蛋白,以纯化的融合蛋白和GST蛋白分别与hela细胞的裂解蛋白进行孵育以沉降(pull down)互作蛋白,SDS-PAGE分离互作蛋白。结果构建了pGEX-4T-2-GJB6(211~261)-GST原核融合表达载体,纯化了融合蛋白及GST蛋白,但在对沉降所得蛋白进行SDS-PAGE分离、检测及比较,未发现明显差异蛋白条带。结论本实验虽未能在hela细胞筛选到GJB6的C-端功能域的互作蛋白,但构建了融合表达载体,纯化了融合蛋白及GST,为进一步GJB6的互作蛋白筛选打下了基础。更多还原

【Abstract】 Objective To screen the interact proteins of the C-terminal functional domain of GJB6 in hela cell for the mechanism study of assembly,trafficking,membrane localization,and formation of normal gap junctions of GJB6.Methods The coding region of 211~261 amino acids of GJB6 gene was amplified from normal human genomic DNA by polymerase chain reaction(PCR).The fusion expression vector of pGEX-4T-2-GJB6(211~261)-GST was constructed.And then,fusion protein and GST were purified.The purified fusion protein and GST were incubated with the disruptive proteins of hela cell to pull down interact proteins.Finally,the interact proteins were separated by SDS-PAGE.Results The fusion expression vector of pGEX-4T-2-GJB6(211~261)-GST was successfully constructed and the fusion protein and GST were successfully purified.But different straps were not found in the "pull down" proteins.Conclusion Although interact proteins of the C-terminal functional domain of GJB6 are not found in the proteins of hela,the fusion expression vector is constructed and the fusion protein and GST are purified.It provides fundament for screening of interact protein of GJB6.更多还原