Genes Expression Of Echinococcus Granulosus Under Oxidative Stress By SSH Technology

Echinococcu granulosus, the cause of cystic echinococcosis, is a small tapeworm that resides in the intestine of carnivore definitive hosts,generally dogs or wolves.The life cycle of E. granulosus consists of four stages: egg or oncosphere, hydatid cyst, protoscolex and the adult worm. Survival of the infectious agent in the mammalian hosts 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, parasites have developed a mechanism of antioxidant defenses, including enzymes that decompose peroxides and superoxide anion (O2-) and compounds that sequester metal ions. Parasites 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.Antioxidants including thioredoxin peroxidas have been identified and characterized in parasitic nematodes e.A new class of antioxidant enzyme has recently been described, which reduces hydroperoxides with thioredoxin as an immediate hydrogen donor. These enzymes are known as thioredoxin peroxidases (TPx). TPx, one of the peroxiredoxin (Prx) gene superfamily, functions as an antioxidant to remove ROS and H2O2 derived from normal cellular metabolism using thioredoxin as the electron donor. TPx have been found in diverse organisms ranging from prokaryotes to mammals. EgTPx may play a role in protecting the parasite from oxidative damage.The larval stages (protoscoleces) are exceptional in having the potential to differentiate in either of two directions. The protoscolex can develop asexually, into the hydatid cyst, or sexually into the adult worm. In vitro culture procedures, PSC were aspirated and pooled from sheep liver hydatid cysts collected from a slaughterhouse in Urumqi, Xinjiang. PSC were collected under sterile conditions from intact cysts, washed three times in phosphate-buffered saline (PBS) prior to digestion in pepsin to release PSC from capsules and remove immature PSC by digestion. After the digestion procedure, the PSC were washed three times with PBS and incubated in glass culture vessels in a liquid DMEM medium placed above a solid-phase base prepared by heating new born calf serum. The solid-phase significantly enhanced PSC growth. After 40 days cultivation, about 10% of the PSC had developed into cysts. In paraffin section of the protoscolex there were many calcareous corpuscle cells, which could provide energy for the protoscolex.The aims of the project are to fully establish and exploit an in vitro culture model that can be used to assess the functions and interactions of a panel of selected E. granulosus genes during differentiation and development of the larval protoscolex stage in an asexual direction to form a hydatid cyst.To isolate specific genes expressed in PSC of E. granulosus under H2O2 stresses, we used suppression subtractive hybridization (SSH) technique with cDNA from PSC treated with H2O2 as tester and cDNA from PSC in normal growth as driver. mRNA was isolated from the tester and driver respectively, and SSH method was employed to analyze the differentially expressed DNA sequence between the two groups. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, the products were subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain DH5α. The cDNA was sequenced and analyzed in GenBank with Blast search. The subtractive library of genes regulated by H2O2 stresses was constructed successfully. The amplified library contained 124 clones, of which all clones had 200-1000 bp inserts. Sequence analysis indicated that 124 clones containing the coding sequences, of which some had homology in the GenBank and other were unknown. The obtained sequences may be target genes regulated by H2O2, which provided foundation for the further identify differentially expressed genes in PSC of E. granulosus exposed to oxidative stress. The cDNA sequence of thioredoxin peroxidase from E. granulosus (EgTPx) was cloned from PSC by RT-PCR and subcloned into the expressive vector pET41-b after sequence analysis。A recombinant plasmid carrying EgTPx gene from was constructed. The resulting recombinant plasmid pET41-b- EgTPx was transformed into the competent cells BL21 and the recombinant plasmid was expressed by IPTG induction.The TPx fusion protein was purified through a Glutathione Sepharose 4B affinity column, and high titer of polyclonal antiserum was obtained by the immunizing the mice using the TPx fusion protein. SDS- PAGE analysis showed that the EgTPx was expressed in E. coli and the relative molecular weight of expressed fusion protein was 54 000Da. ELISA and Western blot indicated the high titer and specifically antiserum produced by protein immune strategy. Construction of pET41-b- EgTPx recombinant plasmid and preparation of the high titer and specifica antiserum were provided as the experimental materials for the EgTPx function analysis.To study the localization of the antioxidant protein–thioredoxin peroxidase (TPx) of E. granulosus (EgTPx) in the protoscolex of the parasite, protoscoleces of E. granulosus were aspirated and pooled from sheep liver hydatid cysts collected from a slaughterhouse in Urumqi, Xinjiang. After digested by pepsin, the sedimented protoscoleces were used for paraffin sections and indirect immunofluorescence staining analysis with polyclonal antibody against rEgTPx. The results indicated that the EgTPx distributed mainly in the tegument, subtegument and calcareous corpuscle cells of the protoscolex. The wide distribution and large sites of EgTPx in the parasite were clearly demonstrated. In the present study, we reported on the cloning, overexpression, immunogenicity, localisation and functional characterisation of E. granulosus TPx, which established a significant clue for further studies of biologic functions and application of TPx protein.In summary, this study could provide primary reference for antioxidant enzymes and genes of E. granulosus. Further studies to elucidate the precise role of antioxidant enzymes may pave the way for considering the enzyme as an alternative target for hydatid disease chemotherapy and present a novel strategy to develop vaccine in human.