title: [Cloning and expression of Toxoplasma gondii autophagy-related protein 3 gene and preparation of its polyclonal antibody].
abstract: OBJECTIVE
  To clone and express autophagy-related protein 3 (TgAtg3) gene of Toxoplasma gondii, and obtain the specific polyclonal antibody against TgAtg3.
  
  
  METHODS
  TgAtg3 cDNA was inserted into prokaryotic expression vector pET28a. After identification, the constructed plasmid pET28a-TgAtg3 was transformed into E. coli Rosetta cells, and induced by special induction medium for expression of the protein. The recombinant protein was purified via Ni-NTA affinity chromatography. Western blotting assay was performed with anti-His tag mouse monoclonal antibody as the primary antibody. Rabbits were immunized with 125 μg purified TgAtg recombinant protein. Each rabbit received 4 immunizations at 2-week intervals with the same dose of antigen. The specific anti-TgAtg3 polyclonal antibody was obtained, and analyzed by Western blotting and indirect immunofluorescence assay (IFA).
  
  
  RESULTS
  pET28a-TgAtg3 plasmid was identified by restriction enzyme digestion, PCR amplification and sequencing. SDS-PAGE and Western blotting analysis showed that the recombinant TgAtg3 protein (about Mr 44,000) was expressed in E. coli Rosetta cells. TgAtg3 protein from tachyzoite lysates was recognized by the specific anti-TgAtg3 polyclonal antibody. IFA assay determined that the specific polyclonal antibody bound to TgAtg3 protein from the cytoplasm of tachyzoites.
  
  
  CONCLUSIONS
  The obtained soluble polyclonal antibody against TgAtg3 can specifically react to the endogenous TgAtg3 protein.
authors.forename: Qian-qian
authors.initials: QQ
authors.lastname: Hua
authors.forename: Xiang-zhi
authors.initials: XZ
authors.lastname: Li
authors.forename: Yu-jing
authors.initials: YJ
authors.lastname: Wan
authors.forename: Di
authors.initials: D
authors.lastname: Chen
authors.forename: Xu-han-hui
authors.initials: XH
authors.lastname: Zhao
authors.forename: Kai
authors.initials: K
authors.lastname: Jiang
authors.forename: Xin
authors.initials: X
authors.lastname: Hu
authors.forename: Chang-wang
authors.initials: CW
authors.lastname: Pan
authors.forename: Feng
authors.initials: F
authors.lastname: Tan
mesh_terms: D000818:Animals; D000906:Antibodies; D015153:Blotting, Western; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D007114:Immunization; D010453:Peptide Synthases; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins; D014122:Toxoplasma
chemical_list: D000906:Antibodies; D018076:DNA, Complementary; D011994:Recombinant Proteins; D010453:Peptide Synthases