Characteristics Of Fasciola Gigantica Thioredoxin Glutathione Reductase And Inhibitors Screening From Furoxan Derivatives

Fascioliasis,is a parasitic disease caused by Fasciola hepatica?F.hepatica?and Fasciola gigantica?F.gigantica?,which reside in the biliary duct and liver.Thioredoxin glutathione reductase?TGR?is a very important antioxidant enzyme in redox system of F.gigantica.Studying and screening inhibitors of TGR can provide a theoretical basis for the development of new anti-Fasciola drugs.In this study,F.gigantica TGR?FgTGR?gene was amplified from the cDNA of F.gigantica by PCR,and fused with the Selenocysteine insertion sequence element?SECIS?of bacteria at end of FgTGR open reading frame.The recombinant prokaryotic expression plasmid pET-28a?+?-FgTGRsec was constructed and co-transformed with the selenoprotein expression engineering plasmid pSUABC into BL21?DE3?cells.The recombinant FgTGR protein with selenocysteine?rFgTGRsec?was obtained,and the pET-28a?+?-FgTGRsec without pSUABC plasmid was also expressed,and the recombinant protein named as r FgTGR.Both of the proteins were purified by his-tag protein purification agarose magnetic beads.The enzymatic activity assays of TrxR,GR and GrxR in rFgTGRsec were established by calculating the obsorbance changes of DTNB and NADPH.In the metal catalytic oxidation system?MCO?,the changes of the supercolied plasmid DNA were observed by DNA agarose gel electrophoresis to evaluate the antioxidant activity of rFgTGRsec.The immunohistochemistry experiment was conducted to observe the distribution of TGR in worm,using sera from mice vaccined by r FgTGR.To abserve the inhibitory effect of 62 new furoxan derivatives to r Fg TGRsec,they were detected by TrxR enzymatic activity assay.rFgTGR and rFgTGRsec were successfully expressed in soluble protein and inclusion body,and the molecular weight were 71 kDa.The results showed that the TrxR enzymatic activity of the purified rFgTGR was 24.28±1.51 U/ml,and that of the purified rFgTGRsec was 54.47±11.66 U/ml.The GR and GrxR enzymatic activitives of the purified rFgTGRsec were 71.14±11.48 U/ml and 11.86±1.88U/ml,respectively.The Michaelis constant of the DTNB substrate was 189.39±52.90?mol/L.Therefore,the insertion of selenium can significantly improve the TrxR activity of FgTGR.It was indicated that selenium played an important role in the function of FgTGR.Supercoiled DNA protection experiment showed that rFgTGRsec could protect supercoiled DNA when the concentration of rFgTGRsec reached at 50?g/ml in the system.However the protein could not protect supercoiled DNA completely even if it was at 600?g/ml.The result indicated that rFgTGRsec owned a role of antioxidant activity.The results of immunohistochemistry showed that TGR was distributed all over the worms,which might reflect the importance of TGR.Among the 62 new furoxan derivatives,LGM-1,LGM-2,LGM-35,ZWJ-19 and CH-33 were screened out and possessed better inhibitory effects.The IC₅₀ values were 6.9 3±1.74?mol/L,15.19±0.80?mol/L,4.5±0.35?mol/L,3.82±0.33?mol/L and 18.54±2.57?mol/L,respectively.This study provided a basis for the further screening inhibitors of FgTGR to develop new anti-Fasciola drugs.