Abstract:

Objective   To construct an eukaryotic vector of human sorbin and SH3 domain containing 1 (hSORBS1) gene and identify the expression and localization of hSORBS1 in osteosarcoma MG-63 cells. Methods   Using GST-hSORBS1 as a template, we obtained human SORBS1 coding sequence by polymerase chain reaction (PCR) amplification and cloned it into the eukaryotic vector 3*Flag. The insert was identified by restriction enzyme digestion and DNA sequencing. 3*Flag-hSORBS1 was transfected into osteosarcoma MG-63 cells. The expression of the recombinant plasmid in MG-63 cells was detected by Western blot. The localization of 3*Flag-hSORBS1 in MG-63 cells was observed with laser scanning confocal microscopy. The hSORBS1 fusion proteins were purified by immunoprecipitation assay. Results   hSORBS1 was constructed into the eukaryotic vector 3*Flag successfully. The expression of 3*Flag- hSORBS1 fusion protein was detected by Western blot, which localization was at the periphery of MG-63 cells. Conclusion   The eukaryotic expression plasmid of hSORBS1 is successfully constructed. The expression of fusion proteins is identified. 3*Flag-hSORBS1 fusion protein is localized at the cell periphery.

Key words: hSORBS1; Osteosarcoma; Eukaryotic expression plasmid; Fusion protein;Immunoprecipitation