[Construction of eukaryotic plasmid of human hCAP gene and the expression and localization of fusion protein].
ABSTRACT To construct the expression plasmid of human c-Cbl-associated protein (hCAP) gene and identify the expression and localization of fusion protein.
Total mRNA was extracted from CV-1 cells, and cDNA was formed by reverse transcription. The hCAP coding sequence was amplified by polymerase chain reaction (PCR) and cloned into pEGFP-C1 plasmid. After the hCAP gene was identified by enzyme digestion and sequencing, the plasmid was transfected into COS-7 cells. The expression of the recombinant plasmid in COS-7 cells was detected by Western blot assay. The localization of pEGFP-hCAP in NIH3T3 cells was observed with laser confocal microscopy.
hCAP was successfully constructed into the pEGFP-C1 expressing vector. The length of the fragment identified by restriction enzyme digestion was 3 879 bp. The expression of pEGFP-hCAP fusion protein with a molecular weight of 169kDa was detected by Western blot. The pEGFP-hCAP fusion protein was mostly localized at the cell periphery of NIH3T3 cells.
The recombinant plasmid of hCAP gene was successfully cloned into eukaryotic expressing vector, and the pEGFP-hCAP fusion protein was mostly localized at the cell periphery of NIH3T3 cells.