pEGFP-N2-ECM1重组质粒的构建及其在人肝癌细胞系SMMC-7721中的表达

Construction of ECM1 in eukaryotic expression vector and its expression in hepatocellular carcinoma cell line SMMC-7721

目的构建细胞外基质蛋白-1(ECM1)稳定表达的SMMC-7721肝癌细胞系。方法应用PCR和DNA重组技术构建pEGFP-N2-ECM1真核表达载体,经酶切、测序鉴定正确后,用脂质体转染SMMC-7721细胞,G418药物筛选稳定转染的细胞系。荧光显微镜检测融合蛋白表达,RT-PCR技术检测ECM1基因表达。结果构建的重组质粒经双酶切及测序分析鉴定,结果证实pEGFP-N2-ECM1构建成功;筛选获得稳定表达ECM1的SMMC-7721细胞。结论成功构建了pEGFP-N2-ECM1的真核表达载体,并在肝癌SMMC-7721细胞中稳定表达,为研究ECM1在肝癌进展中的作用奠定了实验基础。

Objective To establish the hepatocellular carcinoma（HCC） cell line SMMC-7721 with stabilized expression of extracellular matrix protein 1（ECM1）.Methods The complete ECM1 gene amplificated from ECM1 cDNA by PCR was connected into pEGFP-N2 vector by PCR and DNA gene recombinant technique.The recombinant plasmid was detected by restrictive enzyme digestion and gene sequencing analysis;Sequenced right plasmid was transfected into SMMC-7721 cells using lipofectamineTM2000 and ECM1 highly expressed cells were selected with G418.ECM1 gene expression was detected by RT-PCR and the expression of fusion proteins（GFP and ECM1） were observed under the fluorescence microscope.Results The construction of expression vector was accomplished.The SMMC-7721 cell line expressed stably ECM1 which was screened out.Conclusion The eukaryotic expression vector pEGFP-N2-ECM1 is constructed and expressed stably in SMMC-7721 cells,which forms an important basis for the further studies of ECM1 in the HCC progression.