PAM14缺失突变体在哺乳动物细胞中的表达和定位

Localization and expression of human PAM14 deletion mutant gene in mammalian cell lines

目的构建带HA标签(标签序列YPYDVPDYA,源于流感病毒的红细胞凝集素表面抗原决定簇)的PAM14缺失突变体PAM14-N和PAM14-C的真核表达载体,将其分别转染至COS7细胞中观察PAM14-N和PAM14-C蛋白在细胞内的定位,并转染至HEK293T细胞检测其是否表达。方法以含人PAM14全长cDNA序列的质粒pACT2-PAM14为模板,PCR法扩增PAM14-N-HA,BamHⅠ和HindⅢ双酶切后插入真核表达载体pCDNA3.1中,同理构建pCDNA3.1-HA-PAM14-C,将重组表达质粒pCDNA3.1-PAM14-N-HA和pCDNA3.1-HA-PAM14-C分别转染至COS7细胞,在荧光显微镜下观察其细胞定位。转染至HEK293T细胞,提取总蛋白,进行Western blot检测。结果经测序鉴定,成功构建了真核表达载体pCDNA3.1-PAM14-N-HA和pCDNA3.1-HA-PAM14-C,转染后荧光显微镜观察PAM14-N在COS7细胞中主要定位于细胞核,PAM14-C定位不明显;经Western blot检测PAM14-N在HEK293T细胞中能够有效表达,PAM14-C无表达。结论实验结果确定了PAM14功能最小区域及PAM14-N的定位和表达,为进一步了解PAM14的功能奠定了基础。

Objective To clone PAM14 deletion mutant gene into pCDNA3.1 with HA-tagged（Tag sequence YPYDVPDYA,from the influenza virus hemagglutinin surface epitopes）and express this eukaryocyte expression vector in appropriate mammalian cell lines to investigate the localization and the expression of the product of PAM14 deletion mutant gene.Methods PCR methods were used to amplify the open reading frame of PAM14-N-HA from pACT2-PAM14.PAM14-N-HA was cut with BamHⅠ and HindⅢ,and then ligated with purified vector to construct expression vector pCDNA3.1.Accordingly pCDNA3.1-HA-PAM14-C was constructed.The eukaryocyte expression vector was transfected into COS7 cells and HEK293 cells.Fluorescent microscopy and Western blot were performed to detect the localization and expression of PAM14 deletion mutant.Results An eukaryotic expression vector containing PAM14 deletion mutant was constructed.PAM14-N gene was mainly located in the cytoblast of COS7 cells.The position of PAM14-C was not obvious.And the results of Western blot demonstrate that PAM14-N could express stably,while PAM14-C was not stably.Conclusion The results confirm the function minimal domain of PAM14 and the cellular localization and expression of PAM14-N,and provide the basis for further function studies of PAM14 deletion mutant protein.