[Cloning, eukaryotic expression and celluar localization of cysteine and glycine-rich protein 1]

To construct an eukaryotic expression vector of pCMV-FLAG-CRP1 and investigate the cellular localization of cysteine and glycine-rich protein 1 (CRP1) in human embryonic kidney 293T, hepatocellular carcinoma HepG2 and breast cancer ZR75-1 cells.

Human CRP1 gene was obtained from mammary cDNA library by PCR and cloned into the pCMV-Tag2B vector. Human 293T, HepG2 and ZR75-1 cells were transfected with the recombinant plasmid pCMV-FLAG-CRP1; the expression was detected by Western blotting and the cellular localization was observed under a fluorescence microscope.

CRP1 eukaryotic expression vector labeled with FLAG tag was successfully constructed as demonstrated by double enzyme digestion and gene sequencing. Western blotting showed that CRP1 protein was expressed in FLAG-CRP1 transfected 293T, HepG2 and ZR75-1 cells, and fluorescence microscopy revealed that CRP1 protein was localized in both cytoplasm and nucleus of the three cell lines, and the former signal was stronger than the latter.

The eukaryotic expression vector of pCMV-FLAG-CRP1 is successfully constructed, and the cellular location of CRP1 is further identified in the 293T, HepG2 and ZR75-1 cell lines, which will contribute to the further study on the function of CRP1.