title: [Cloning, Expression and Immunodiagnostic Evaluation of the Fasciola gigantica Thioredoxin Peroxidase].
abstract: OBJECTIVE
  To immunoscreen the gene encoding thioredoxin peroxidase (TPx) from a cDNA library made from adult Fasciola gigantica worms, clone and express the gene, and evaluate the immunodiagnostic value of TPx recombinant protein.
  
  
  METHODS
  The A ZAP cDNA library was immunoscreened with pooled serum of fascioliasis gigantica patients. The obtained positive clones were sequenced and analyzed by multiple sequence alignment. The full-length (rFgTPx) and N-termianal truncated (rFgTPx_nt) sequence of FgTPx was subcloned into prokaryotic plasmid pET28a(+) with a non-fusion expression technique, respectively. The recombinant proteins of rFgTPx and rFgTPx_nt were purified by His-bind affinity column (Ni-NTA). rFgTPx and rFgTPx_nt were used in indirect ELISA to test the antibody response of the serum samples. Sera of 27 fascioliasis gigantica patients, 15 patients with schistosomaisis japonica, 15 clonorchiasis sinensis patients, and 32 healthy donors were tested by using the recombinant protein based ELISA.
  
  
  RESULTS
  The TPx recombinant proteins were obtained through expression, purification and renaturation, the relative molecular mass of rFgTPx and rFgTPx_nt were Mr 30,000 and Mr 26,000, respectively. The total diagnostic coincidence rate, sensitivity and specificity of rFgTPx_nt-based ELISA was 87.6% (78/89), 66.7% (18/27), and 96.8% (60/62), respectively. The cross reaction with Schistosoma japonicum and Clonorchis sinensis was 0 and 1/15 for rFgTPx_nt, respectively. Before and after treatment, A450 value of the serum samples from fascioliasis patients was 0.233 ± 0.088 and 0.129 ± 0.072, respectively (t = 4.27, P < 0.01).
  
  
  CONCLUSIONS
  The gene encoding TPx is expressed in the prokaryotic expression system. The recombinant protein shows proper sensitivity and high specificity for the serodiagnosis of Fasciola gigantica infection.
authors.forename: Yue-qi
authors.initials: YQ
authors.lastname: Wang
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhou
authors.forename: Na
authors.initials: N
authors.lastname: Cheng
authors.forename: Mu-xin
authors.initials: MX
authors.lastname: Chen
authors.forename: Lin
authors.initials: L
authors.lastname: Ai
authors.forename: Yu-hua
authors.initials: YH
authors.lastname: Liu
authors.forename: Jian-guo
authors.initials: JG
authors.lastname: Zhang
authors.forename: Jia-jun
authors.initials: JJ
authors.lastname: Luo
authors.forename: Xue-nian
authors.initials: XN
authors.lastname: Xu
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D003003:Clonorchiasis; D003004:Clonorchis sinensis; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D005209:Fasciola; D005211:Fascioliasis; D015870:Gene Expression; D015723:Gene Library; D006801:Humans; D054464:Peroxiredoxins; D010957:Plasmids; D011994:Recombinant Proteins; D012549:Schistosoma japonicum; D016415:Sequence Alignment; D012698:Serologic Tests
chemical_list: D011994:Recombinant Proteins; D054464:Peroxiredoxins