title: DKK1 eukaryotic expression plasmid and expression product identification.
abstract: We constructed the human dickkopf 1 (DKK1) eukaryotic expression plasmid and expressed, purified, and identified its expression product. We extracted cancer cells from cervical cancer tissue, followed by extraction of mRNA. Reverse transcription-polymerase chain reaction was conducted to obtain DKK1 gene fragments. Using these fragments, we prepared the recombinant plasmid pCMV-HA2/DKK1. The recombinant plasmid was restriction enzyme-digested and sequenced, and using liposome vectors, was transiently transfected into Free-Style 293-F cells (serum-free medium). DKK1 protein was detected by western blotting. The amplification product showed the expected size. Restriction enzyme digestion and sequence analysis showed that the recombinant plasmid was PCMV-HA2/DKK1. The expression product was verified properly by western blotting using an anti-DKKI antibody. The successful cloning of the DKKI gene and expression of DKKI protein will be useful for studying the biological activity of tumorigenesis.
authors.affiliation: Department of Clinical Laboratory, Yangzhou First People's Hospital, Yangzhou, Jiangsu, China baogy_fml@163.com.
authors.forename: G Y
authors.initials: GY
authors.lastname: Bao
authors.affiliation: Department of Clinical Laboratory, Yangzhou First People's Hospital, Yangzhou, Jiangsu, China.
authors.forename: K Y
authors.initials: KY
authors.lastname: Lu
authors.affiliation: Department of Clinical Laboratory, Yangzhou First People's Hospital, Yangzhou, Jiangsu, China.
authors.forename: S F
authors.initials: SF
authors.lastname: Cui
authors.affiliation: Department of Clinical Laboratory, Yangzhou First People's Hospital, Yangzhou, Jiangsu, China.
authors.forename: L
authors.initials: L
authors.lastname: Xu
mesh_terms: D063646:Carcinogenesis / Q000235:genetics*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000235:genetics*; D056890:Eukaryota / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D005822:Genetic Vectors; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics
chemical_list: C501126:DKK1 protein, human; D036341:Intercellular Signaling Peptides and Proteins; D012333:RNA, Messenger