Shen Tao, Li Yan, Yang Li-qing, Liang Shuang, Ba Gen, Fu Qin
Department of Orthopedic Surgery, Shengjing Hospital of China Medical University, Shenyang, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2012 Mar;28(3):237-9.
To construct an eukaryotic expression vector GFP-hArgBP2 and identify the expression and localization of hArgBP2 in osteosarcoma MG-63 cells.
Using pcDNA3.1-hArgBP2 as a template, we obtained human ArgBP2 coding sequence by polymerase chain reaction (PCR) amplification and cloned it into the eukaryotic expression vector. The insert was identified by restriction enzyme digestion and DNA sequencing. GFP-hArgBP2 was transfected into osteosarcoma MG-63 cells and examined by Western blot. The localization of GFP-hArgBP2 in MG-63 cells was also observed with laser scanning confocal microscopy. hArgBP2 protein was purified by immunoprecipitation assay.
hArgBP2 was successfully constructed into the expressing vector pEGFP-C1. The length of the fragment identified by restriction enzyme digestion was 1 935 bp. The expression of GFP-hArgBP2 fusion protein with a molecular weight of 97 000Da was detected by Western blot and pulled down by GFP antibody, and its localization was in the cytoplasm and perinucleus in MG-63 cells.
The recombinant plasmid of hArgBP2 gene was successfully constructed. The expression of GFP-hArgBP2 fusion protein was identified and pulled down by GFP antibody. GFP-hArgBP2 was mainly localized in the cytoplasm and perinucleus of MG-63 cells.
