人食管癌相关基因4原核表达载体构建及其重组蛋白对食管癌EC-18细胞周期的影响  

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

作　　者：李林蔚 王文玉 李晓燕 史祖宣 高天慧 Li Linwei;Wang Wenyu;Li Xiaoyan;Shi Zuxuan;Gao Tianhui(Department of Oncology,Henan Provincial People 's Hospital,Henan University People 's Hospital,Zhengzhou University People's Hospital,Zhengzhou 450003,China)

机构地区：[1]河南省人民医院河南大学人民医院郑州大学人民医院肿瘤科,郑州450003

出　　处：《肿瘤研究与临床》2018年第11期721-724,共4页Cancer Research and Clinic

基　　金：国家自然科学基金(U1304817);郑州市科技攻关项目(141PPTGHG298).

摘　　要：目的构建人食管癌相关基因4(ECRG4)原核表达载体,表达纯化ECRG4重组蛋白,验证ECRG4重组蛋白的生物学功能。方法利用DNA重组技术构建ECRG4重组蛋白原核表达载体。转化大肠杆菌,表达纯化ECRG4重组蛋白,十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)分析ECRG4重组蛋白纯度。用磷酸盐缓冲液(PBS)和10μg/ml ECRG4重组蛋白分别处理食管癌EC-18细胞48h,流式细胞术检测肿瘤细胞周期的变化。结果SDS-PAGE分析显示获得了高纯度的ECRG4重组蛋白,成功构建了ECRG4蛋白原核表达载体。PBS对照组与ECRG4重组蛋白组G1期细胞比例分别为(60.4±2.1)%、(71.6±1.8)%(t=25.695,P=0.002),S期细胞比例分别为(24.6±1.4)%、(16.5±1.0)%(t=36.905,P=0.001),G2/M期细胞比例分别为(15.0±1.1)%、(11.9±0.8)%(t=6.471,P=0.023),提示ECRG4重组蛋白能诱导EC-18细胞G1期阻滞。结论成功构建ECRG4原核表达载体,ECRG4重组蛋白在食管癌中具有活性生物学功能。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 (10μg/ml)for phosphate buffer (PBS)48h 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.

关 键 词：食管癌相关基因4 重组蛋白 细胞周期 

分 类 号：R735.1[医药卫生—肿瘤]