title: [Prokaryotic Expression and Immunoreactivity Analysis on Profilin of Toxoplasma gondii].
abstract: OBJECTIVE
  To clone and express the profilin (PRF) gene of Toxoplasma gondii, and analyze the immunoreactivity.
  
  
  METHODS
  Total RNA was extracted from tachyzoites of T. gondii RH strain. The coding region of TgPRF was amplified with a pair of specific primers. PCR product was digested with double restriction enzyme and ligated into pET30a(+) vector. The recombinant pET30a(+)-TgPRF plasmid was transformed into E. coli DH5α with positive clones confirmed by the double restriction enzyme digestion, PCR and sequencing. The correct plasmid was transformed into E. coli BL21 and induced by IPTG. The expressed proteins were purified with Ni-NTA affinity chromatography and analyzed by SDS-PAGE. Western blotting with rabbit anti-T. gondii serum was used to analyze its antigenicity.
  
  
  RESULTS
  The product of RT-PCR was with 492 bp. pET30a-TgPRF was confirmed by the double restriction enzyme digestion, PCR and sequencing. SDS-PAGE analysis showed that the expressed product was a soluble protein with a relative molecular weight of 35,000. Western blotting assay revealed that rTgPRF was recognized by rabbit anti-T. gondii serum.
  
  
  CONCLUSIONS
  TgPRF gene has been expressed in prokaryotic expression system and shows immunoreactivity.
authors.forename: Fei
authors.initials: F
authors.lastname: Yuan
authors.forename: Zhuan-zhuan
authors.initials: ZZ
authors.lastname: Liu
authors.forename: Bo
authors.initials: B
authors.lastname: Zhang
authors.forename: Jian-ping
authors.initials: JP
authors.lastname: Cao
authors.forename: Kui-yang
authors.initials: KY
authors.lastname: Zheng
authors.forename: De-guang
authors.initials: DG
authors.lastname: Wang
mesh_terms: D015153:Blotting, Western; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D015870:Gene Expression; D005822:Genetic Vectors; D010957:Plasmids; D016133:Polymerase Chain Reaction; D051304:Profilins / Q000276:immunology*; D015800:Protozoan Proteins / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology; D014122:Toxoplasma
chemical_list: D051304:Profilins; D015800:Protozoan Proteins; D011994:Recombinant Proteins