title: [Bioinformatics analysis on CK8 full length coding sequence and eukaryotic expression in SMMC7721 cells].
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  The eukaryotic expression vector containing the CK8 gene was successfully constructed and expressed, which provides a basis for the study for biological function of CK8.
authors.affiliation: Department of Immunology and Microbiology, Xi'an Medical School, Xi'an 710021, China.
authors.forename: Meng
authors.initials: M
authors.lastname: Xun
authors.forename: Si-Hai
authors.initials: SH
authors.lastname: Zhao
authors.forename: Chun-Xia
authors.initials: CX
authors.lastname: Cao
authors.forename: Xin
authors.initials: X
authors.lastname: Xue
authors.forename: Ming-Ming
authors.initials: MM
authors.lastname: Shao
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.forename: Kun
authors.initials: K
authors.lastname: Xu
authors.forename: Yong-Lie
authors.initials: YL
authors.lastname: Chu
mesh_terms: D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D019295:Computational Biology; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D053533:Keratin-8 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection
chemical_list: C426203:KRT8 protein, human; D053533:Keratin-8; D011994:Recombinant Proteins