Preliminary Study On The Biological Characteristics Of Thioredoxin Glutathione Reductase Of Orientobilharzia Turkestanicum

Orientobichiasis is a zoonotic parasitic disease caused by six species of Orientobilharzia.In China,the main epidemic species is Orientobilharzia turkestanicum.Adult worms were mainly parasitic in the mesenteric veins and livers of goats and cattle.The lesions mainly occur in the liver tissue,but the disease is less harmful to humans,and usually cause cercarial dermatitis in humans.However,the Orientobilharzia turkestanicum live in the blood vessels of host such as cattle and goats.Orientobilharzia turkestanicum needs oxidoreductase to resist reactive oxygen free radicals(ROS)produced by the host immune system or metabolism,because the parasite live in the blood vessels of host.However,O.turkestanicum lacks two independent systems of Trx and Grx.Instead,a multifunctional enzyme thioredoxin glutathione reductase(TGR)system,which possess thioredoxin reductase(Trx R),glutathione reductase(GR)and glutaredoxin(Grx)three enzyme activities.In view of this unique characteristic,the research on the parasitic worm TGR highly attract attention by parasitologists.In this study,we established and maintained the life cycle of O.turkestanicum under laboratory conditions.At the same time,different commonly used experimental animals were tried as the final hosts of Orientobilharzia turkestanicum.We successfully maintained the life cycle of O.turkestanicum using goats as the final host.However,adult worms could survive in rabbits,rats and guinea pigs,and no adult worm could be found in mice and Microtus fortis.O.turkestanicum could be collected from rabbits at 180-day post infection.Then,PCR technology was used to amplify the entire open reading frame(ORF)sequence of the TGR gene from the c DNA of O.turkestanicum,and the selenocysteine insertion element(SECIS)contained in Escherichia coli was inserted into the end of the ORF of Ot TGR.The recombinant prokaryotic expression plasmids p ET-28a(+)-Ot TGR and p ET-28a(+)-Ot TGRsec were constructed to express the recombinant proteins,r Ot TGRsec and r Ot TGR.The two proteins were purified by His-tag protein purification agarose magnetic beads,respectively.r Ot TGR and r Ot TGRsec were successfully expressed in E.coli.r Ot TGR was expressed in soluble and inclusion body,while r Ot TGRsec was mainly expressed in soluble protein.In E.coli(BL21,DE3),the expression amount of r Ot TGR was higher than r Ot TGRsec.Polyclonal antibodies against to Ot TGR was prepared,and Western blotting and immunohistochemistry experiments were carried out to observe the distribution and expression of Ot TGR in the worms.The results of immunohistochemistry experiment showed that r Ot TGR was mainly distributed in the tegument of the parasite,and it was also distributed in some other tissues.We established TrxR,GR,Grx three enzyme activity detection system of r Ot TGRsec,and detected and calculated Trx R,GR and Grx activities,The enzymic activity results of r Ot TGRsec showed that the Trx R enzyme activity was 12.30±1.63 U/m L,the GR enzyme activity was 8.83±3.15 U/m L,and the Grx enzyme activity was 0.31±0.07 U/m L,respectively.Moreover,the inhibitory effect of Furoxan derivatives on Trx R enzyme activity of r Ot TGRsec was detected.As the Furoxan derivatives,LGM-35,ZWJ-19 and CH-33 were all possessed inhibitory effect on the Trx R activity of r Ot TGRsec.Finally,RNA interference technology was used to interfere the expression of Ot TGR gene in vitro.The interference effects of different si RNA molecules on the gene were compared and analyzed,and the effect of LGM35 on O.turkestanicum was evaluated in vitro.The results of RNA interference showed that among the 6 si RNAs,they displayed different interference effects,and the highest interference rate could reach 46.1%.Western blot results showed that r Ot TGRsec existed two obvious bands,and mass spectrometry analysis showed that both of them were the protein.The compound LGM-35 revealed the killing effect on O.turkestanicum.However,the effect was relatively weaker compared with that in Schistosoma japonicum.This study maintained the life cycle of the O.turkestanicum under laboratory conditions,and conducted a preliminary study of the biological function of the TGR gene,for the follow-up of the laboratory research of O.turkestanicum and the development of new targets with Ot TGR.Laid the foundation for the anti-O.turkestanicum.