title: [Expression and characterization of thioredoxin glutathione reductase of Schistosoma japonicum].
abstract: OBJECTIVE
  To prepare the recombinant thioredoxin glutathione reductase of Schistosoma japonicum (SjTGR) with biological activity.
  
  
  METHODS
  The open reading frame DNA sequence of SjTGR was fused with a bacterial-type selenosysteine insertion sequence (SECIS) element by PCR to form a chimeric gene. The chimeric gene was subcloned into expression plasmid pET-41a to construct a recombinant plasmid SjTGR-pET-41a. Then the recombinant plasmid SjTGR-pET-41a was co-transformed into E. coli BL21 with plasmid pSU ABC. The SjTGR protein was expressed by inducing with IPTG. The recombinant SjTGR was purified from expression products by affinity chromatography with an adenosine 2', 5'- diphosphate agarose column. The polyclonal anti-serum against recombinant SjTGR was obtained by immunizing mice with purified SjTGR. The native TGR in S. japonicum was evidenced by using Western blotting. Thiorendoxin reductase (TrxR) activity, glutathione reductase (GR) activity and gluaredoxin (Grx) activity of recombinant TGR were analyzed according to the biochemical method.
  
  
  RESULTS
  The chimeric gene of SjTGR with a bacterial-type selenosysteine insertion sequence (SECIS) element was constructed successfully. The bacteria containing the recombinant plasmid SjTGR-pET-41a could express the soluble SjTGR by inducing with IPTG at static growth stage for 24 h at 24 degrees C. The expressed products of plasmid pSU ABC could promote the integration of selenocysteine and increase the yield of selenoprotein. The result of Western blotting showed that the polyclonal antiserum against recombinant SjTGR could recognize the native TGR in S. japonicum adult worms. The enzymatic assay indicated that SjTGR was a multifunctional enzyme with activities of TrxR, GR and Grx.
  
  
  CONCLUSIONS
  The recombinant SjTGR with biological activity is expressed successfully, which lays the foundation for further study on the function and applied values of SjTGR.
authors.affiliation: Jiangsu Institute of Parasitic Diseases, Key Laboratory on Technology for Parasitic Disease Prevention and Control, Ministry of Health, Wuxi 214064, China.
authors.forename: Li-Jun
authors.initials: LJ
authors.lastname: Song
authors.forename: Chuan-Xin
authors.initials: CX
authors.lastname: Yu
authors.forename: Shu-Ying
authors.initials: SY
authors.lastname: Xie
authors.forename: Xu-Ren
authors.initials: XR
authors.lastname: Yin
authors.forename: Chun-Yan
authors.initials: CY
authors.lastname: Qian
authors.forename: Jie
authors.initials: J
authors.lastname: Wang
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.forename: Xue-Dan
authors.initials: XD
authors.lastname: Ke
mesh_terms: D000818:Animals; D015870:Gene Expression; D015801:Helminth Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D051379:Mice; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012549:Schistosoma japonicum / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D012554:Schistosomiasis japonica / Q000469:parasitology
chemical_list: D015801:Helminth Proteins; D009097:Multienzyme Complexes; D009247:NADH, NADPH Oxidoreductases; C466433:thioredoxin glutathione reductase