Cloning, Expression And Identification Of Thioredoxin Peroxidase Gene From Echinococcus Granulosus

Echinococcosis is a cosmopolitan zoonosis caused by adult or larval stages of cestodes belonging to the genus Echinococcus, which is badly harm for human health and stockbreeding production. Now the therapy of Cystic Echinococcosis(CE) in humans is mainly according to surgery or drug treatment,but the surgical operation has a large of damnification for human body and can not ensure that they will not infection again. Meanwhile, patients with CE who take drug treatment may bring fearful ill reaction. So it is impossible to cure CE completely. Larval infection (hydatid disease, hydatidosis) is characterized by long-term growth of metacestode (hydatid) cysts in the intermediate host and easy to diffuse, we have no effective prevent measure as yet. so the early diagnosis and prevent for most human cases of CE is become more and more important. Because it is difficult to diagnose CE with traditional etiology method, often use physical imaging methods and immunodiagnosis as assistant measure.The immunodiagnosis with its low expense, simple operation and high specificity become important measure in CE diagnosis. But there is a lack of standard and specific antigen which interfere the experiment sensitivity and result determination. Therefore, finding new candidate gene of E. granulosus, their expression, purification, and identification of their immunological characters is necessary .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 H2O2 derived 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 cestodes. It has been demonstrated that protoscoleces(PSC)of Echinococcus granulosus(hydatid cysts)can metabolize hydrogen peroxide in vitro, but, given that neither catalase nor glutathione peroxidase activities were detectable, EgTPx may play a role in protecting the parasite from oxidative damage. so the TPx was considered to be the hotspot in present research.In the present study, thioredoxin peroxidase(TPx) gene from Echinococcus granulosu(sEg) was cloned by RT-PCR, the prokaryotic and eukaryotic recombinant plasmid was constructed and expressed. Then the immunological and antioxidant characters of the recombinant thioredoxin peroxidase in E. coli and Pichia pastoris expression system was identified respectively. Providing valuable candidate gene to develop vaccine for preventing Eg.Firstly, total RNA was extracted from PSC of cysts of sheep. The specific primers were designed according to published nucleotide sequence of NCBI/GenBank database. The TPx gene of Eg was amplified by RT-PCR and insert into pET-41b vector and transfer into E.coil BL21 to construct the expressed recombinant plasmid pET-41b-EgTPx. Then sending the recombinant plasmid pET-41b-EgTPx to company for sequencing. The homology of EgTPx gene was analyzed using DNAman software and NCBI/BLAST database. Then the genetically engineered bacteria pET-41b- EgTPx were induced by IPTG, the expression products were analyzed by SDS- PAGE and then the recombinant protein EgTPx/GST(rEgTPx) was purified using Glutathione Sepharose 4B affinity column. As a result, a gene sequence of 582 bp has been successfully amplified by RT-PCR .Comparing the DNA and deduced amino acid sequence with the published EgTPx gene sequence of Eg to revealed 99.83%and 100% homology respectively. The SDS- PAGE analysis showed that the EgTPx was expressed in E. col.i and the relative molecular weight (Mr) of expressed fusion protein was 54 000. Secondly, the purified rEgTPx was used as antigen to immunize mice and obtain the purified serum for testing the biological activity of rEgTPx by western blot and ELISA. Western blotting analysis show that the antiserum was specific against the rEgTPx protein and PSC antigen, and the titer of the antiserum detected by ELISA was 1 :25 6000 .Thirdly, prokaryotic expression systerm is lack of correctly fold the foreign protein and perform other post-translational modifications mechanism, so E. coli expressed proteins also tend to retain their amino-terminal methionine, which may affect protein stability. The methylotrophic yeast Pichia pastoris, as an eukaryotic expression system, has been a favorite system for expressing heterologous proteins due to its unexampled advantages. In this paper, we attempted to produce EgTPx with biologically activity using P. pastoris expression system . A cDNA fragment from PSC was cloned by RT-PCR and inserted into expression vector pPIC9K, designated as pPIC9K-EgTPx. The recombinant vector was linearized by restriction enzyme Bgl II and transformed into GS115 by electroporation.Three recombinant strains containing multiple copies were obtained by G418 resistance screening and then they were identified by PCR analysis. Under the control of the promoter AOX1, the gene was induced with 1% methanol to secret EgTPx into culture medium. The culture supernatant was analyzed by SDS-PAGE and Western blot, which showed that the protein of EgTPx has a molecular mass of 22 KDa and could bind to EgTPx specific polyclonal antibodies. An in vivo assay showed that the secreted EgTPx could significantly increased the tolerance and survival of the cells existing in high concentrations of H2O2 comparing with controls. The successful cloning and expression of EgTPx in Pichia pastoris has laid a foundation for its further application.