Cloning,Expression And Characetrization Of Arginase From Schistosoma Japonicum

Schistosomiasis caused by infection with Schistosoma japonicum cercariae was one of the mostprevalent tropical parasitic zoonosis, which is serious harm to health of people and animal in China. Itwas worthwhile to screen new drug target and develop the new candidate vaccine for further controllingand prevention of the disease. Arginase as a metabolic enzyme responsible of hydrolyzing arginine toproduce ornithine and urea, plays an important role in cell proliferation, collagen deposition and energymetabolism, but also affects the body’s immune response by competing against with nitric oxidesynthase (NOS) for expendable substrate arginine to adjust the product of NO.In our lab，schistosoma japonicum arginase (SjARG) was identified in the previous research oftegument and surface protein proteomics in schistosomula. To further clarify the characteristics andbiological function of SjARG, its gene was cloned by PCR with primer based on schistosome arginasereference sequence. The SjARG open reading frame (ORF) of1095bp, encoding364amino acids, is79.7%homologous with Schistosoma mansoni arginase (SmARG). There were two divalent Mn2+binding sites observed in the enzymatic center by3D modeling, similar to SmARG active sites. Therecombinant plasmid pET-28a-SjARG was constructed, and transformed into competent E.coil/BL21toobtain the engineeing bacteria which was capable of producing recombinant SjARG protein (rSjARG)with molecular weight (MW) of43kDa. Its antigenicity was confirmed by Western blotting analysis. Acomparative quantitative PCR analysis showed that the copy number of the SjARG gene transcript incercariae was highest, about20-100folds higher than other investigated stages, and the transcriptionlevel in42-day-old female worms was significantly higher than that in males. Immunolocalizationanalysis revealed that SjARG was mainly distributed in tegument, parenchyma and concentrated ingenital area of the42d adult male and female worms.Enzymatic analysis system of rSjARG was constructed, and enzyme activity assay revealed thatrSjARG arginase activity was about502U/mg and had the optimum pH of10.2and temperature of37℃. The Km value of the enzyme was0.0827mol/L. Mn2+can significantly increase the arginaseactivity of rSjARG, while Cu2+and Zn2+decreased the activity. No enzymatic activity can be measuredafter incubation at4℃and70℃. At the present of Mn2+, heat-activation was not essential for rSjARG.L-cysteine had an intensive impact on the activity of the enzyme, and ornithine and lysine showedweaker effects. The depression of reducing agent such as dithiothreitol (DTT) and reduced glutathionewas correlated with its concentration. S-(2-boronoethyl)-L-Cysteine (BEC) inhibited theSjARG-activity in a dose dependent manner and2(S)-Amino-6-boronohexanoic acid (ABH) was thestrongest inhibitor of rSjARG. Both Nw-hydroxy-L-Arginine (NOHA) and Nw-hydroxy-nor-L-Arginine(nor-NOHA) can significantly inhibit the rSjARG activity, and their inhibitory effect was dependent onthe substrate concentration of arginine. The BALB/c mice administrated with NOHA by tail veininjection were induced20.23%(P<0.05) and27.47%(P<0.05) decrease in the adult worm burden aswell as31.50%(P<0.05) and17.28%(P<0.05) decrease in liver egg counting post infection, comparedto the PBS control group in the two independent trials. Treatment of Schistosoma japonicum cercariae with NOHA obtained52.00%(P<0.05) and50.84%(P<0.01) worm reduction and31.50%(P<0.05) and55.25%(P<0.01) eggs reduction in the two independent mouse infection experiments, compared to theuntreatment control.In two independent trials, BALB/c mice were immunized with rSjARG combining ISA206adjuvantand resulted in partial protective efficacy,28.94%(p<0.01) and36.35%(p<0.05) worm reduction,33.64%(p<0.01) and42.67%(p<0.01) liver eggs reduction, respectively. ELISA assay showed thatvaccinated mice were induced significant increased level of specific IgG, IgG1and IgG2a antibody, buta reduced IgG1/IgG2a ratio. Bio-Plex analysis revealed that serum cytokines levels of IL-2, IL-12andIL-10in were considerable increased by immunization.In summary, the results suggested that SjARG may have the potential as a new vaccine candidate anddrug target against schistosome, and provided a basis for in-depth study of the biological function of theenzyme.