Construction Eukaryotic Express Vector Of Human S100a13 Gene And Its Effect On Cell Cycle Of Hepg2

Objective:To obtain eukaryotic express vector of human S100A13 gene and then to investigate the effects of exogenous S100A13 overexpression on the cell cycle of human hepatocellular liver carcinoma cell line HepG2.METHODS:S100A13 open reading frame sequence were amplified by PCR from pGEM-T-S100A13 vectors, which conserved in our lab, then were subcloned into pcDNA3.2/V5-topo vector and sequenced. The eukaryotic expression plasmid pcDNA3.2/V5-S100A13 was transfected into HepG2 cells. The subcellular localization of S100A13 protein was detected by immunoflurescence and the effects of S100A13 on cell cycle progression were measured by flow cytometry after 48 hours of transfecting.RESULTS:S100A13 gene was identified to be inserted to pcDNA3.2/V5-topo vector correctly. Indirect immunofluorescence indicated S100A13 protein was uniformly localized in the cytosol and nucleus of HepG2-S100A13-V5 cells, while was mainly localized in nucleus of HepG2-V5 cells and HepG2 cells.Both S phase proportion and G2/M phase proportion were significantly higher in HepG2-S100A13-V5 cells than in HepG2-V5 and HepG2 cells [(24.90±0.66)% vs. (17.11±0.23)% and (16.7±0.36)% at S phase, P＜0.01; (12.19±0.14)% vs. (7.26±0.23)% and (7.51±0.28)% at G2/M phase, P＜0.01].CONLUSION:The eukaryotic expression vector containing human S100A13 gene was successfully constructed and expressed in HepG2 cells.Overexpression exogenous S100A13 gene could promote cell cycle progression of HepG2 cells from G0/G1 phase to S and G2/M phase. In HepG2 cells, exogenous S100A13-V5 fusion protein localized in the cytoplasm,while endogenous S100A13 localized in the nucelus.