Expression, Enzymatic Activity Of Cysticercus Cellulose DUTPase And Its Structural Prediction

Cysticercosis caused by Taenia solium is an important zoonotic disease.It is particularly prevalent in central America, northern South America and some parts of Africa and Asia. The disease, one of six urgently-eradicating diseases issued by WHO, has not only brought about a great economic loss, but also endangered the public health. Vaccines and drugs which have many defects are primary ways to prevent cysticercosis. The design of new vaccine and drug becomes a critical element for developing successful control strategies. The ubiquitous enzyme dUTPase, which is essential in both eukaryotes and prokaryotes, catalyzes the hydrolysis of dUTP to dUMP and PPi and prevents mismatch in DNA duplication. The dUTPase has been studied as a novel and valid target against many diseases.The gene encoding dUTPase from Cysticercus cellulose (CY) was first cloned. The nucleotide homology is 100% between oncosphere and c.cellulose of the T.solium. In order to further study the biological functions, the CY dUTPase was cloned into pET28 vector and expressed in BL21(DE3) and purified by Ni2+-IDA-Sepharose. At the same time, the pET28 vector as a control was expressed by the same way. The SDS-PAGE result showed the recombinant CY dUTPase (21KD) had not been contaminated by host's dUTPase. dUTPase catalyzes the hydrolysis of dUTP to dUMP and PPi. The activity of dUTPase was determined by OD values at 575nm of the complex of PPi and molybdate solution. The quantity of the recombinant dUTPase for catalyzing lnmol dUTP every minute was 21.3095μg. One unit of enzyme activity was 21.3095μg.The specific activity of the recombinated dUTPase was 46.929U/mg and its activity was enhanced by Mg²⁺ and inhibited by EDTA.The amino acid homology of dUTPase from different species was more than 60%. The 3D protein structure model of c.cellulose dUTPase was generated by SWISS-MODEL in its first approach mode. The model was refined by SPDBV and DSViewer,and the quality of mode was analyzed by WHAT IF and ProSac program. There were 27 pockets in the mode analysised by CASTp program. It showed the same structure between the 26 pocket and homotrimer dUTPase which the crystal structure was obtain in PDB. Some conserved residues such as Ala(83),Tyr(90) and the glycine-rich C-terminal tails were consensus with the other homotrimer dUTPases. The CY dUTPase we presumed belonged to the homotrimer which was generated by SymmDock and superposition with 3D structure of Human dUTPase. In conclusion we have made the model of CY dUTPase successfully, which provids the material foundation for a novel drug design against Cysticercosis.The siRNA sites were designed by program online, a homologous and complementary sequence against U6 promoter 3' terminal was added to the 3' end of shDNA (short hairpin DNA) which was synthesized by IDT Co. The U6 promoter was obtained by PCR from mouse genome. For generating template used in PCR for preparing SEC (siRNA expression cassettes), the U6 promoter and shDNA against CY dUTPase were extended together. The SECs were yielded by overlapping extension single-step PCR with universal primers. The method of Semi-Quantitative RT-PCR, in which the clone pAT5 gene was used as an internal standard, was generated to detect the effect of RNAi. Preparing for further studies on RNAi in T.solium cells and development of new antisense drugs against Cysticercosis.