title: [Construction and expression of an eukaryote vector of 14-3-3 protein in Toxoplasma gondii].
abstract: OBJECTIVE
  To construct and express the eukaryotic expression vector of 14-3-3 protein of Toxoplasma gondii RH stain.
  
  
  METHODS
  The structure and physicochemical property of 14-3-3 protein were predicted by bioinformatics analysis tools. The desired gene fragment was amplified from total RNA in T. gondii RH strain by RT-PCR, and sub-cloned into pcDNA3.0 to construct recombinant plasmid pcDNA3.0/14-3-3. After PCR confirming, double restriction enzyme digestion and DNA sequencing, the eukaryotic expression vector pcDNA3.0/14-3-3 was transfected into HeLa cells and the target protein was detected by Western blotting.
  
  
  RESULTS
  The prediction of its gene sequence and amino acid sequence suggested that the 14-3-3 protein was acid soluble protein with five conserved regions, existing as homo- or hetero-dimers. The amplified gene fragment was about 800 bp, and the inserted fragment in pcDNA3.0/14-3-3 was 801 bp by sequencing, which had 99% homology to the 14-3-3 gene sequence of T. gondii in GenBank (Accession No. AB012775.1). Western blotting showed that there was more 14-3-3 protein expressed in the pcDNA3.0/14-3-3-transfected HeLa cells than not-transfected and mock transfected cells. Its relative molecular mass (Air) was about 30 000.
  
  
  CONCLUSIONS
  The eukaryotic expression vector pcDNA3.0/14-3-3 is constructed and expressed in eukaryotic cells.
authors.affiliation: Department of Pathogen Biology, Shandong University School of Medicine, Jinan 250012, China.
authors.forename: Min
authors.initials: M
authors.lastname: Sun
authors.forename: Shen-Yi
authors.initials: SY
authors.lastname: He
authors.forename: Guang-Hui
authors.initials: GH
authors.lastname: Zhao
authors.forename: Hua
authors.initials: H
authors.lastname: Cong
authors.forename: Huai-Yu
authors.initials: HY
authors.lastname: Zhou
authors.forename: Qun-Li
authors.initials: QL
authors.lastname: Zhao
authors.forename: Min
authors.initials: M
authors.lastname: Meng
mesh_terms: D048948:14-3-3 Proteins / Q000235:genetics*; D019295:Computational Biology; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D010957:Plasmids; D014122:Toxoplasma / Q000235:genetics; D014162:Transfection
chemical_list: D048948:14-3-3 Proteins