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目的:构建人细胞角蛋白8(CK8)全长CDS序列真核表达载体,并转染人肝癌细胞SMMC7721,为研究CK8的生物学功能提供细胞模型。方法:RT-PCR扩增CK8全长CDS,克隆入pMD18-Tsimplevector质粒,鉴定正确后,亚克隆入质粒pEGFP-C1,构建真核表达载体pEGFP-CK8。脂质体介导转染SMMC7721细胞,荧光显微镜、realtimePCR和Westernblot检测CK8的表达。生物信息学软件分析CK8理化特性、信号肽及功能位点等。结果:PCR、酶切和测序结果均证实重组质粒含有人CK8全长CDS序列,转染实验表明CK8在SMMC7721细胞中发生了高表达。结论:成功地构建了人CK8全长CDS真核表达载体,并使其在SMMC7721细胞中获得高表达,为研究CK8的生物学功能奠定了实验基础。
Objective: To construct eukaryotic expression vector of human cytokeratin 8 (CK8) full length CDS sequence and transfected into human hepatocellular carcinoma cell line SMMC7721 to provide a cell model for studying the biological function of CK8. Methods: The full-length CDS of CK8 was amplified by RT-PCR and cloned into pMD18-Tsimplevector plasmid. After identified correctly, it was subcloned into plasmid pEGFP-C1 to construct eukaryotic expression vector pEGFP-CK8. Liposome-mediated transfection of SMMC7721 cells, fluorescence microscope, realtimePCR and Western blot detection of CK8 expression. Bioinformatics software analysis CK8 physical and chemical properties, signal peptides and functional sites. Results: The full length CDS sequence of human CK8 was confirmed by PCR, restriction enzyme digestion and sequencing. Transfection experiments showed that CK8 was overexpressed in SMMC7721 cells. CONCLUSION: The eukaryotic expression vector of human CK8 full length CDS was successfully constructed and was highly expressed in SMMC7721 cells, which laid the experimental foundation for studying the biological function of CK8.