title: [Gene-cloning, expression and immunoreactivity detection of Toxoplasma gondii uridine phosphorylase].
abstract: OBJECTIVE
  To predict the physicochemical properties and antigenic epitopes of Toxoplasma gondii uridine phosphorylase (TgUPase), clone, and express TgUPase gene, and analyze its immunoreactivity.
  
  
  METHODS
  The physical and chemical characters and specific epitopes of TgUPase protein were predicted by bioinformatics software tools. Total RNA was extracted from RH strain T. gondii tachyzoites. A pair of specific primers was designed according to the open reading frame of TgUPase gene (GenBank Accession No. DQ385446.1). RT-PCR product was digested with restriction enzyme and ligated into a pET-30a(+) vector. The recombinant plasmid pET-30a(+)-TgUPase was transformed into E. coli DH5alpha and the positive clones were selected by colony PCR and confirmed by double restriction enzyme digestion and sequencing. The constructed pET-30a(+)-TgUPase was then transformed into E. coli BL21(DE3) and induced with IPTG for expression. The expression product was analyzed through SDS-PAGE followed by Coomassie blue staining. Western blotting assay with His primary antibody and human anti-T. gondii serum was used to confirm the expression of rTgU-Pase and detect its immunoreactivity.
  
  
  RESULTS
  Bioinformatics prediction results showed that rTgUPase protein was 303 amino acids in length with a predicted molecular mass of M, 33 042.9, and this soluble protein had three potential T/B cell epitopes. The product of RT-PCR was 921 bp. Colony PCR, double restriction enzyme digestion and DNA sequencing confirmed that the recombinant plasmid pET-30a(+)-TgUPase was constructed. SDS-PAGE showed that bacteria containing recombinant plasmid pET-30a(+)-TgUPase expressed a soluble protein of His-TgUPase (about Mr 38,000) after being induced with IPTG. The recombinant protein reacted positively with His primary antibody and human anti-T. gondii serum by Western blotting analysis.
  
  
  CONCLUSIONS
  The recombinant plasmid pET-30a (+)-TgUPase is constructed and the soluble rTgUPase shows immunoreactivity.
authors.forename: Li-Tian
authors.initials: LT
authors.lastname: Yin
authors.forename: Jian-Jiang
authors.initials: JJ
authors.lastname: Zhu
authors.forename: Run-Hua
authors.initials: RH
authors.lastname: Li
authors.forename: Hai-Long
authors.initials: HL
authors.lastname: Wang
authors.forename: Ya-Qing
authors.initials: YQ
authors.lastname: Li
authors.forename: Guo-Rong
authors.initials: GR
authors.lastname: Yin
mesh_terms: D000906:Antibodies; D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes; D004926:Escherichia coli; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins; D014122:Toxoplasma / Q000201:enzymology / Q000276:immunology*; D014543:Uridine Phosphorylase / Q000276:immunology* / Q000378:metabolism
chemical_list: D000906:Antibodies; D000939:Epitopes; D011994:Recombinant Proteins; C025991:Thomsen-Friedenreich antibodies; D014543:Uridine Phosphorylase