title: [Cloning and expression of actin gene of Toxoplasma gondii].
abstract: OBJECTIVE
  To clone and express the actin gene of Toxoplasma gondii, and analyze the immunoreactivity of the recombinant protein.
  
  
  METHODS
  Total RNA was extracted from tachyzoites of RH strain of T. gondii. The open reading frame of TgACT gene was amplified with a pair of specific primers which were designed according to the coding sequence of TgACT gene (Accession No. XM_002369622.1). The RT-PCR product was cloned into the prokaryotic expression pET-30a (+) vector. The recombinant pET30a-TgACT plasmid was transformed into E. coli DH5alpha. The positive clones were selected through the colony-PCR and confirmed by the double restrict enzyme digestion and sequencing. The correct pET30a-TgACT plasmid was transformed into E. coli BL21(DE3) and induced by IPTG. The expressed proteins were analyzed by SDS-PAGE. Western blotting assay was performed with anti-poly-histidine tag (anti-His) antibody or rabbit anti-T. gondii serum.
  
  
  RESULTS
  The product of RT-PCR was with 1 100 bp. The recombinant plasmid pET30a-TgACT was confirmed by colony-PCR, double restriction enzyme digestion and sequencing. SDS-PAGE results showed that the target protein was expressed in E. coli BL21(DE3) in the form of inclusion bodies with a rough molecular weight of 49 000. The purified soluble protein was obtained by using denaturation, renaturation and purification. Western blotting revealed that rTgACT can be recognized by anti-His antibody and rabbit anti-T. gondii serum.
  
  
  CONCLUSIONS
  The recombinant plasmid pET30a-TgACT has been successfully constructed, and the recombinant protein TgACT is produced in E. coli and maintains specific immunoreactivity.
authors.forename: Run-Hua
authors.initials: RH
authors.lastname: Li
authors.forename: Hai-Xia
authors.initials: HX
authors.lastname: Hao
authors.forename: Hai-Long
authors.initials: HL
authors.lastname: Wang
authors.forename: Xiao-Li
authors.initials: XL
authors.lastname: Meng
authors.forename: Jin-Yan
authors.initials: JY
authors.lastname: Shen
authors.forename: Guo-Rong
authors.initials: GR
authors.lastname: Yin
mesh_terms: D000199:Actins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D003001:Cloning, Molecular; D015870:Gene Expression; D005822:Genetic Vectors; D010957:Plasmids; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014122:Toxoplasma / Q000235:genetics* / Q000378:metabolism
chemical_list: D000199:Actins; D015800:Protozoan Proteins; D011994:Recombinant Proteins