Construction of prokaryotic expression vector with human esophageal cancer related gene 4 and the effect of recombinant protein on the cell cycle of EC-18 in esophageal carcinoma / 肿瘤研究与临床

ABSTRACT

Objective To construct the prokaryotic expression vector of human esophageal cancer related gene 4 (ECRG4), to purify the recombinant ECRG4 protein and to verify the biological function of the recombinant ECRG4 protein. Methods DNA recombination technology was utilized to construct the ECRG4 protein prokaryotic expression vector. The recombinant ECRG4 protein was purified with the transformation of escherichia coli. Then the purity of the recombinant ECRG4 protein was examined by using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Furthermore, esophageal cancer EC-18 cell line was treated by recombinant ECRG4 protein (10 μg/ml) for phosphate buffer (PBS) 48 h respectively, and the tumor cell cycle change was examined by using flow cytometry. Results SDS-PAGE analysis showed that the high purity of recombinant ECRG4 protein was obtained and the ECRG4 protein prokaryotic expression vector was successfully constructed. The cell proportion of G1 phase in PBS control group was lower than that in the recombinant ECRG4 protein group [(60.4 ±2.1) ％ vs. (71.6 ±1.8) ％; t= 25.695, P= 0.002]. The cell proportion of S phase in PBS control group was higher than that in the recombinant ECRG4 protein group [(24.6±1.4) ％ vs. (16.5±1.0) ％; t= 36.905, P= 0.001]. The cell proportion of G2/M phase in PBS control group was higher than that in the recombinant ECRG4 protein group [(15.0 ±1.1) ％ vs. (11.9 ±0.8) ％; t=6.471, P=0.023], which indicated that the recombinant ECRG4 protein could induce the G1 phase arrest of EC-18 cells. Conclusion The ECRG4 protein prokaryotic expression vector is successfully constructed. And the recombinant ECRG4 protein has an active biological function in esophageal carcinoma.