| Babesiosis is a kind of tick-borne blood protozoosis. The parasite replicates in the host’s red blood cells, causing fever, anemia, hematuria and even death of human and animals. Babesia microti, the most severe pathogenic agent of Babesia spp, can infect with a variety of animal, and human. Elderly people and splenectomized or immunocompromised persons usually suffer with severe infections or death.Protozoan, multiplied in erythrocytes of mammalian hosts, have to counteract the toxic effects of reactive oxygen and nitrogen species(ROS, RNS) that could potentially lead to major oxidative DNA damage and lipid peroxidation. Thus, in order to counteract the oxidative stress and maintain a redox balance, parasites employ a thioredoxin system, which includes the nicotinamide-adenine dinucleotide phosphate(NADPH), the thioredoxin(Trx)and the NADPH dependent disulphide oxidoreductase,namely thioredoxin reductase(TrxR). The Trx system has been extensively studied in Plasmodium falciparum, while the enzyme system has been rarely studied in babesia that causes babesiosis.Therefore, to understand the molecular mechanism of the Trx system in babesia and drug targets screening, we carried out this research on the key enzyme TrxR from B.microti.In the present study, there are the following four contents:1. The transcriptome sequences of B.microti at erythrocyte stage were analyzed and we found an important thioredoxin reductase in the redox metabolic pathways. We obtained the sequence of full-length cDNA of BmiTrxR by RACE. BmiTrxR cDNA was 1766 bp(GenBank,No. KU845266)and contained a single open reading frame with 1659 bp that encoded a polypeptide with 553 amino acids. Molecular weight of the predicted protein was 58.4 kDa and an isoelectric point of 6.95. The predicted signal peptide cleavage site was between 18 and 19 amino acids. The putative protein had a thiol-disulfide redox active center(-CVNVGC-) in its N-terminus and the C-terminus contained a conserved GCGGGKCG sequence, which are the catalytic domain of TrxR. BLAST analysis of the predicted protein against all NCBI non-redundant databases revealed significant similarity with members of TrxR from other species. The amino acid sequence of Bmi TrxR was 58% identical to Babesia bovis TrxR, 53% identical to Plasmodium falciparum TrxR.2. Full-length Bmi TrxR was subcloned into pET-28 a expression vector and successfully expressed as a His-tag fusion protein in E.coli BL21. The recombinant protein had a molecular weight of 58.4kDa, which is in agreement with the expected size predicted from the amino acid sequence. After affinity chromatography column purification, the recombinant protein was purified and a polyclonal antibody in mice was prepared,3. Western blotting indicated that polyclonal antibodies in mice could recognize the native Bmi TrxR in B.microti which is 58.4 kDa. IFAT showd the specific fluorescence seemed to localize in the cytoplasm of B.microti merozoites.4. Enzyme activity of the purified recombinant Bmi TrxR was analyzed and the activity of Bmi TrxR against NADPH-dependent disulfide reductase using DTNB as substrate was significant. Inaddition, auranofin as the inhibitor, could abrogated the enzyme activity of Bmi TrxR completely at 12.5μM,In conclusion, this study has cloned the full-length cDNA of the TrxR of B.microti and completed expression and purification of the recombinant protein, confiremed location of TrxR, verified the NADPH-dependent disulfide reductase activity of Bmi TrxR. Research results lay the foundation for better illuminating the molecular mechanism of TrxR antioxidative system in babesia and also provide clues for discovery of the parasite-resistant drug target. |
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