Objective: 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.
Methods: 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.
Results: 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.
Conclusion: 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.