In Vitro Expression And Tissue Distribution Of Taenia Solium DUTPase

The essential enzyme dUTP pyrophosphatase (dUTPase) is exquisitely specific for dUTP and is critical for the fidelity of DNA replication and repair. In this study, the taenia solium dUTPase gene was amplificated from hatched and activated oncospheres by RT-PCR and spliced leader-based PCR, and was directed cloning to pGEM-T Easy plasmid, sequencing and sequence analysis. Then, this gene was ligated into pET-28a plasmid and pGFP-C1 plasmid, constructed prokaryotic and eukaryotic expressed reorganization plasmid, and transformed into E.coli BL21(DE3) cells and BHK cells. The prokaryotic expressed protein was analyzed by SDS-PAGE protein electrophoresis and western-blot method. Eukaryotic expression was observed by fluorescence microscope directly. Eventually, through cutting gel and electroeluting to purify prokaryotic expressed protein, and using the highly purified protein to immune Kunming mice to obtain anti-dUTPase polyclonal antibody, detecting by agar bridge. Proglottid of taenia solium and Cysticercus cellulosae were fixated by formalin to paraffin section, staining by HE and immunohistochemistry simultaneously, and negative serum was used control in immunohistochemistry.The results showed that an 571bp fragment of taenia solium dUTPase cDNA with an ORF of 447bp was obtained. The fragment included 5ËŠ-termina spliced leader and coded 149 amino acids. The dUTPase of taenia solium and Schistosom japonicum was in the same clade. pE-dUTPase and pG-dUTPase expressed plasmid were constrcted successfully. The pE-dUTPase recombinant transformants were both expressed interest protein with IPTG or non-IPTG induction; The expressed products could be recognized by onchosphere swine positive serum, showed the fusion protein have certained antigenicity. Transfected 48 hours, green fluorescence of the pG-dUTPase recombinant transformants can be observed by fluorescence microscope. Obtained highly purified protein by cutting gel and electroeluting. Antibody titer of anti-dUTPase was 1:32. Immunohistochemistry positive signal showed dUTPase expressed mainly in scolex and strobila wall, in strobila essence vacuolus was less expressed, and nearly no expressed in neck segment, less expressed in gaps of Cysticercus cellulosae parenchymatous tissue. These finding and experimental data will help us to further study of structure of dUTPase molecule and protein fuction.