Cloning、Expressioin And Biochemical Characterizations Research Of1-deoxy-d-xylulose-5-phosphate Reductoisomerase(DXR) In Toxoplasma Gondii

Toxoplasma gondii is a kind of parasitic protozoa, which is distributed in the worldwide, and brings great harm to human life and Animal Husbandry production.So far, there is no breakthrough in Immune Control and Prevention, and the effective Clinical anti-Toxoplasma drugs is unavailable, searching for the effective, low toxicity new drugs against Toxoplasma gondii is still one of the important directions of Toxoplasma gondii research. Isoprenoids play important worles as compoments of structure cholesterol,steroid hormones in mummels,carotenoids in plants,and abiquinones in all livery organism.Recently,the genomic data analysis has revealed that a novel plant-like.The pathway of2C-methyl-D-erythritol-4-Phosphate(MEP) is widely present in the Isoprenoid Anabolic way of plants, bacteria and protozoa.However, this pathway does not exist in Human and animal bodies. DXP reductoisomerase(DXR) is the scond key enzymes in the MEP pathway, which is a potential target for the design of new Antibacterial and antiprotozoal drugs. In this paper,the gene for coding the1-deoxy-D-xylulose-5-phosphate reductoisomerase of Toxoplasma gondii (TgDXR) was cloned and Prokaryotic expressed. Enzyme kinetics and inhibition kinetics features were analyzed,and The experiments include the following contents:Firstly,the TgDXR ORF was cloned using a pair of specific Primers which is desiged accoding the predicted gene sequence deposed in the ToxoDB. The whole length of TgDXR gene is1542bp,coding a513amino acid composition, molecular weights55.7kD, Analysis by Signal P3.0revealed that TgDXR possess a leader peptide fragment including a signal peptide of22amino acids (initiated from N-terminal) and an apicoplast targeted transit peptide of43amino acids, molecular weights48.6kD Multiple sequences alignment showed TgDXR have identity of36-63%in amino acid composition with other model organisms, and TgDXR secondary structure contents45.1%Helix,22.9%Sheet,8.7%Turn and23.3%Coil.By the means of DNA recombination, encoding region of TgDXR(TgDXR1: ORF;TgDXR2:Region of TgDXR without N-terminal signal peptide and transit peptide)was sucloned into pET-32a(+) and pMAL-c2x vector, and transformed into E. coli Rosseta(DE3) for expressing respectively.The recombinant vectors were induced by IPTG at different temperature respectively. Comparison of these expressions indicate that only the fragment encoding mature protein without N-terminal signal peptide and transit peptide were expressed.The inclusion bodies and solution of pMAL-c2x-TgDXR were higher than the pET-32a(+)-TgDXR.The solution of pMAL-c2x-TgDXR was purified by Amylose Resin column purification systemThe enzymatic characterization of TgDXR was studied. The recombinant form (r TgDXR) expressed in E. coli Rosseta(DE3) was able to catalyse the substrate of DXP and NADPH,with Mn2+existence. However, it can oxidize NADPH with a Km of36.3±3.2μM and DXP with a Km11.5±0.8μM. Function of recombinant TgDXR could be inhibited by Fosmidomycin and the IC50for Fosmidomycin was8.8±0.4μM, which suggest that this enzyme maybe explored as a potential and novel drugs target.