Expression And Enzyme Activity Analysis Of The Two Key Enzymes To The Isoprenoid Pathway In Babesia Orientalis

Babesia orientalis is one of the parasites belonging to apicomplexan phylum in order of Piroplasmida transmitted by vector Rhipicephalus haemaphysaloides to water buffalo which is the specific host.The disease can be spread for generation of years on adult stage and is most prevalent in central part of China principally at provinces of Hubei,Hunan,Guanxi,Yunan and some others provinces,causing serious economic loss in water buffalo livestock industry.Nowadays there is not yet an effective recombinant vaccine against diseases caused by parasites apicomplexan pathogens including Babesia orientalis,that obviously make a big challenge for developing novel chemotherapeutic component available for market in the perspective of these diseases control.In fact,the isoprene metabolic pathway essential for parasites life metabolism,different to mevalonate pathway in mammals,is present in apicoplast structure of apicomplexan parasites and has been identified as a promising chemotherapeutic target.Therefore,the key enzymes involved in that metabolic pathway are important and has a great significance for analysis.The whole genome sequencing of Babesia orientalis which is not yet published was completed in our laboratory and several genes have been identified and accurately annotated.1-deoxy-D-glucose-5-phosphate synthase(DXS)and1-deoxy-D-glucose-5-phosphate reductoisomerase are the first and second key rate limiting enzyme in the MEP pathway which is a potential drug target for anti protozoal drugs.In this work,we have successfully cloned the two genes cited above from B.orientalis genome library,and bioinformatic analysis,prokaryotic expression and functional analysis were carried out.The experiment was divided in two sections:(1)In this part,two enzymes DXS and DXR of Babesia orientalis were obtained successfully from the genome database of Babesia orientalis and the sequences were submitted on NCBI regarding the highest homology of nucleotide and amino acid.Two specifics primers were designed based on the homologous sequences by using gDNA and cDNA as template to amplify the target gene and inserted in the cloning vector pEASY-Blunt for sequencing to confirm the identity.Bioinformatic tools were used for the two proteins analysis.The result has shown that BoDXS is a 2094 bps nucleotide size in length encoding a protein of 697 amino acid without any introns,and has a predicted signal peptide at N-terminal region in position amino acid 23.The amino acid sequence of BoDXS contains high conserved regions with its homologues in others apicomplexan genera.BoDXR the full sequence is a 1554 bps length in size encoding for a protein of 457amino acid with a predicted signal peptide at N-terminal region,but contrary to BoDXS,BoDXR contains introns.(2)The second part has consisted mainly on construction of two prokaryotic expressions vector:pET32a-BoDXS and pET32a-DXR.The immunization was carried out to obtain rabbit BoDXS polyclonal antibody and mouse DXR polyclonal antibody and western blot was used to verify the immunogenicity of these two proteins.The recombinant Babesia orientalis protein DXS and DXR were identified respectively as a 91 kDa in size and 68 kDa mostly expressed in inclusion body,but a low expression of BoDXS was observed in low temperature and low speed conditions.The proteins in inclusions bodies were extracted and purified,and the protein activity was restore for enzyme activity determination.Western blot analysis result has shown that both in Babesia orientalis whole antigen and Babesia orientalis positive serum were able to detect the native protein confirming that the expression of fusion protein in vitro and in vivo is identical.These results provide a scientific basis for the application of protein latter.(3)The recombinant BoDXR was analyzed by enzyme kinetic method.The reaction rate was measured by reducing fluorescence value under OD 340 mm condition.The result has indicated that BoDXR was able to oxidize NADPH and catalyze the formation of DXP by MEP.BoDXR has a Km value of 360 ± 34 μM for NADPH and a Km value of 620 ± 59 μM for DXP.The maximum reaction rate of BoDXR is 13.25 ± 2.6 μM · min-1·μg-1.