Molecular Cloning, Expression And Protein Activity Analysis Of Thioredoxin Peroxidase Gene From

Schistosomiasis,the zoonotic infectious parasitic disease,is one of the major public health problems which greatly threatening human health in China. Oncomelania hupensi is the sole intermediate host of Schistosoma japonicum.Therefore, O.hupensis plays a key role in the transmission of the S.japonicum. Survival of the infectious agent in the host requires adaptive mechanisms,such as producing antioxidant enzymes against reactive oxygen species(ROS) generated by the host immune responses.ROS are generally considered to be cytotoxic because high concentrations of ROS can have seriously deleterious effects on membrane lipids,nucleic acids and proteins.To avoid the oxygen toxicity,hosts have developed a mechanism of antioxidant defenses,including enzymes that decompose peroxides and superoxide anion(O2-)and compounds that sequester metal ions.Hosts have adapted to considerable oxidative stress by synthesizing high levels of antioxidant enzymes and,in many cases,expressing them at the host-parasite interface.The levels of these defense enzymes are correlated with the survival of a number of parasitic helminths in host tissues. The TPx family is a large family of antioxidant proteins universally found in all living organisms,from prokaryotes to eukaryotes.TPx functions as an antioxidant to remove the reactive oxygen species(ROS)and H2O2derived from normal cellular metabolism using thioredoxin as the electron donor.TPx have also been implicated in oxidative signalling mechanisms regulating apoptosis,cell differentiationand cell proliferation. In parasites,antioxidant enzymes have been extensively characterized in parasitic nematodes and trematodes,where they play a key role in protecting these organisms from the potentially damaging effects of ROS and host-activated leukocytes,but few studies have been carried out on O.hupensis. TPx may play a role in protecting the snail from oxidative damage.so the TPx was considered to be the hotspot in present research.In the present study, thioredoxin peroxidase (TPx) full length gene from O.hupensis was cloned by RACE-PCR,the prokaryotic recombinant plasmid was constructed and expressed.Then the antioxidant characters of the recombinant thioredoxin peroxidase in E.coli expression system was identified.The total RNA was extracted from O.hupensis.The specific primers were designed according to published nucleotide sequence of NCBI/GenBank database.The TPx full length gene from O.hupensis was cloned by RACE-PCR and submitted to the NCBI. The homology of TPx gene was analyzed using NCBI/BLAST database. TPx gene was inserted into pET-28a vector and transfer into E.coil BL21to construct the expressed recombinant plasmid pET-28a/TPx.Then sending the recombinant plasmid pET-28a/TPx to company for sequencing.The homology of TPx gene was analyzed using DNAman software and NCBI/BLAST database.Then the genetically engineered bacteria pET-28a/TPx was induced by IPTG,the expression products were analyzed by SDS-PAGE and then the recombinant protein TPx/His was purified using Ni chromatography and NTA cation exchange chromatography.As a result,the full length gene sequence of992bp has been successfully amplified by RACE-PCR. The recombinant plasmids were identified by enzyme digestion and DNA sequencing.SDS-PAGE analysis showed that the recombinant protein was the fusion body with the relative molecular weight of about27000Da,and the target protein.Secondly, peroxidase activity of O.hupensis TPx protein was assayed with purified TPx fusion proteins and H2O2with the presence (+DTT) or absence (-DTT) in a concentration dependent manner. The remaining amount of H2O2was measured spectromertrically and calculated as the percentage of H2O2removal by TPx.Values are the means±SD of three similar experiments. Protection of super-coiled DNA cleavage by recombinant O.hupensis TPx was analyzed in metal-catalyzed oxidation (MCO). The pUC18DNA with MCO(3.3mmol/L DTT and16.5μmol/L Fecl3) system and was added in recombinant TPx with a concentration gradient (2.5,5.0and10.0μg/ml) Peroxidase activity of O.hupensis TPx protein showed that the protein can removal the H2O2with the presence of DTT in vitro. Protection of super-coiled DNA cleavage by recombinant O. hupensis TPx in MCO showed that TPx proteion was able to protect super-colied DNA from the damage by MCO in vitro.Thirdly, Relative abundance of TPx transcripts in O.hupensis were detected by real-time Q-RT-PCR in O.hupensis following no exposure(Ohr) or exposure to S. japonicum miracidia for5hr,10hr,24hr or48hr.The fold change of expression of the transcripts was calculated by comparison between unexposed and exposed snails witnin each snail stock by the comparative Ct method.The result showed that differences in the levels of TPx transcript induction following in fection,with the transcript up-regulated in snails during the early phase(10h)of infection compared to unexposed snails in which it was down-regulated within the late time period. The result indicated that TPx gene could be involved in the infection of S. japonicum miracidia into snail and might be played an role in the stress..