PAM14的细胞转染表达和定位

Expression and localization of PAM14 in transfected cell

目的构建带GFP标签的PAM14表达载体,将其转染至HEK293T细胞检测其表达,转染至COS7细胞观察PAM14蛋白在细胞内的定位。方法以含人PAM14全长cDNA序列的质粒pACT2-PAM14为模板,用PCR方法扩增PAM14全长序列,然后插入带GFP标签的载体pCDGFP中构建表达载体pCDGFP-PAM14,将构建的质粒转染至HEK293T细胞,提取总蛋白,进行Western-blot检测;转染至COS7细胞,在荧光显微镜下观察其细胞定位。结果成功构建了表达载体pCDGFP-PAM14,此表达载体在HEK 293T细胞中能够有效表达,转染表明PAM14在COS7细胞主要在核内表达,胞质内也有少量表达。结论实验结果为进一步了解PAM14的功能提供了一定的基础。

Objective To Clone PAMI4 gene into pCDGFP vector and express this vector in appropriate mammalian cell lines to investigate the expression and localization of the product of PAM14 gene. Methods The full length cDNA fragment of PAM14 from pACT2-PAM14 vector was used to amplify with PCR ,then the fragment was inserted into pCDGFP vector to construct expression vector pCDGFP-PAM14. Its expression was detected in HEK 293T cells transfected with GFP-tagged PAM14 gene with western-blot and the location of PAM14 protein defined in COS7 cells transfected with GFP-tagged PAM14. Results The expression vector encoding PAM14 protein could express egiciently in HEK 293T cell line, and the product of PAM14 gene was found to be localized both in the nucleus and cytoplasm, mainly in the nucleus in COS7 cells. Conclusion The results provide the basis for further function studies of PAM14 protein.