Bioinformatics analysis on CK8 full length coding sequence and eukaryotic expression in SMMC7721 cells

• Published in: Xi bao yu fen zi mian yi xue za zhi Vol. 27; no. 2; p. 123
• Main Authors: Xun, Meng, Zhao, Si-Hai, Cao, Chun-Xia, Xue, Xin, Shao, Ming-Ming, Li, Wei, Xu, Kun, Chu, Yong-Lie
• Format: Journal Article
• Language: Chinese
• Published: China 01.02.2011
• Subjects: Cell Line, Tumor; Cloning, Molecular; Computational Biology; Gene Expression Regulation; Genetic Vectors - genetics; Genetic Vectors - metabolism; Humans; Keratin-8 - chemistry; Keratin-8 - genetics; Keratin-8 - metabolism; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Transfection
• ISSN: 1007-8738

To construct an eukaryotic expression vector containing the coding region of human full length cytokeratin 8 gene and to detect its expression in SMMC7721 cells. CK8 cDNA was amplified by RT-PCR and cloned to pMD18-T simple vector. After confirming the sequence, the cDNA was inserted into pEGFP-C1 and the positive clone pEGFP-CK8 was obtained. The recombinant plasmid was transfected into SMMC7721 cells with Lipofectamine(TM);2000 and the expression was detected by fluorescence microscope, real time PCR and Western blot. The physical-chemical properties, signal peptide and functional motifs were predicted by the bioinformatics software. PCR, restriction enzyme digestion and DNA sequencing showed that the recombinant plasmid contained the coding region of full length CK8 gene. Observation under fluorescence microscope and the results of real time PCR and western blot indicated CK8 was over-expressed in SMMC7721 cells. The eukaryotic expression vector containing the CK8 gene was successfully constructed and expr