Schistosoma Japonicum Thioredoxin Glutathione Reductase (sjtgr) Preparation And Properties Of The Research

Schistosomiasis is a severse parasitic disease, affecting millions of peoples in China, and hasbecome a worldwide public health problem. Currently, schistosomiasis infections mainly dependon chemical medicine treatment of praziquantel, they must be retreated on an annual orsemiannual basis for the rapid re-infection. Therapies based on just a single compound are riskybecause of the high probability to cause resistance. In fact, schistosoma mansoni strains displayingpraziquantel susceptibility have already been detected in laboratory. Hence, it is the urgent task fordeveloping of new antischistosomal chemotherapy drugs based on an available drug targets.The study showed that in S. japonicum, sepcialized TrxR and GR enzymes are absent, andreplaced by a multifunctonal enzyme, thioredoxin glutathione reductase （TGR）. ThioredoxinGlutathione Reductase from Schistosoma Japonicum, SjTGR, plays a key role in maintaining theredox balance within the cell of Schistosoma japonicum. Research showed that RNA interferenceof the SjTGR gene expression will result in the death of schistosome, and the protease meet thisstandard as a drug target protein. In this study, we expressed and isolated the recombinant proteinsof SjTGR and SEC-SjTGR, and the enzyme assays were determined. These studies lay a solidfoundation for the research of inhibitors screen based on this target protein.Firstly, the gene of thioredoxin glutathione reductase from Schistsoma japonicum （SjTGR）was cloned to the expression vector of pET-28a. Then the recombinant plasmid pET28a-SjTGR.was transformed into Escherichia coli expression host BL21. Recombinant protein of SjTGR wasinduced by IPTG and captured by the Ni ion affinity chromatography and further purified withDEAE ion affinity chromatography. SDS-polyacrylamide gel electrophoresis was used to detectthe protein and determined the molecular weight of enzyme. The SEC-SjTGR, the native form ofSjTGR containing a carboxylterminal sequence of GCUG is a selenoprotein and the―U‖isselenocysteine（SEC）. For the expression of SEC-SjTGR, we introduced a variant bacterial-typeselenoprotein insertion sequence （SECIS） element followed the3’ sequence of SjTGR gene andformed the plasmid of SjTGR-SECIS-pET28a. SjTGR-SECIS-pET28a and pSUABC plasmidwere co-transformed into BL21. The pSUABC plasmid contains selA, selB, selC gene （encondingselenocysteine synthase, SELB and tRNASEC, resepectively）. Expression of recombinantselenoprotein was induced by IPTG and captured by the methods mentioned above.Secondly, using ultraviolet-visible spectrophotometer, the activities of thiorendoxin reductase（TrxR）、glutathione reductase （GR）、gluaredoxin （Grx） and their respective Michaelisconstant of recombinant SjTGR were determined at25℃with the substrates of5,5’-dithiobis（2-nitrobenzoic acid）（DTNB）, triphosphopyridine nucleotide （NADPH）, glutathione oxidized （GSSG）, glutathione reduced （GSH）, and β-hydroxyethyl isulfide （HED）.The trials results showed that the SEC-SjTGR was a multifunctional enzyme with activities ofTrxR, Grx and GR and the values of enzyme activities were6.440.51μmol min^-1mg^-1（DTNB）,138.975.43μmol min^-1mg^-1（HED）,12.550.49μmol min^-1mg^-1（GSSG）,respectively.；And the SjTGR don’t have TR and GR activity, but has the Grx activity and theactivity of257.089.52μmol min^-1mg^-1（HED）.The Km values of different substrates ofSEC-TGR were1.46μMNADPH（DTNB）,18.8618μMNADPH（GSSG）; The Km valuesof different substrates of SjTGR were1061.3885μMNADPH（HED）,524.8337μM（HED）.