硒蛋白S过表达载体的构建及其在肝癌细胞SMMC7721中的表达鉴定

Construction of overexpression vector of SELS gene and expression in liver cancer SMMC7721 cells

目的构建硒蛋白S基因(SELS)真核表达载体pCMV-SELS,转染肝癌SMMC7721细胞,实现SELS在SMMC7721中的成功过表达。方法通过基因克隆技术将SELS基因的完整ORF插入真核表达载体pCMV-3tag-3a(+),得到阳性真核表达质粒pCMV-SELS,将其瞬时转染到肝癌SMMC7721细胞中,利用RT-PCR与Western blot方法验证SELS的过表达水平。结果成功构建了真核表达质粒pCMV-SELS,将其瞬时转染SMMC7721细胞后24 h,收集细胞进行RT-PCR和Western blot检测,结果显示:与阴性对照组pCMV-3tag-3a(+)转染组相比,实验组pCMV-SELS细胞中SELS的mRNA和蛋白表达水平均有显著升高,两组比较具有统计学差异(P<0.05)。结论成功构建真核表达载体pCMV-SELS,并实现了SELS基因在SMMC7721细胞中的过表达。

Objective To construction an eukaryotic expression vector pCMV-SELS and observe its expression in liver cancer SMMC7721 cells, and to provide experimental basis for study on the relationship between SELS gene and liver cancer. Methods The liver cancer SMMC7721 cells were transfected transiently with the constructed eukaryotic expression vector pCMV-SELS. The expression of SELS gene in the untransfected SMMC7721 cells, the SMMC7721 cells transfected with pCMV-3tag- 3a（＋）-SELS and the SMMC7721 cells transfected with pCMV-3tag-3a（＋）（control） were detected by RT-PCR and Western blot. Results The eukaryotic expression vector pCMV-3tag-3a（＋）-SELS was successfully constructed. The expression of SELS gene in the cells transfected with pCMV-SELS was significantly increased compared with the cells transfected with pCMV-3tag-3a（＋） and the untransfected SMMC7721 cells（P0.05）, The Western blot results showed that the expression of SELS gene in the cells transfected with pCMV-SELS was significantly increased compared with the cells transfected with pCMV-3tag-3a（＋） and the untransfected SMMC7721 cells. Conclusion The eukaryotic expression vector pCMV-3tag-a（＋）-SELS is successfully constructed. The SELS gene highly expression in the SMMC7721 cells transfected with expression vector.