Molecular And Subcellular Location In Sporozoites Of 2C-Methyl-D-erythritol 2,4-cyclodiphosphate Synthase Of Eimeria Tenella

Coccidiosis is an important avian disease which damages seriously to the poultry production.The strategies of controlling this important economically disease rely heavily on chemoprophylaxis.Due to increasing emergency of drug-resistant strains, exploring the new target for anti-coccidials had been eesiential.Non-mevalonate isoprenoid biosynthesis is an important anabolism pathway,which exsits generally in plant,bacterium and some primitive organism.The MEP pathway had proved to be exsited in plasmodium according to the data,and the new antimalarial,fosmidomycin, had been developed based on this pathway as the target.In this paper,the ORF encoding the 2C-Methyl-D-erythritol 2,4-cyclodiphosphate synthetase of Eimeria tenella(EtMEC),one of the key enzymes in isoprenoid biosynthesis,was predicted by comparative genomics and bioinformatics.There were two major experiments in this study:(1) Bioinformatic predictive analysis and cloning of EtMEC.There was partial encoded MECS sequence in E.tenalla Contig₀003528 to be found by searching the E.tenalla genomic database with P.falciparum MECS amino acid array(Gene Bank Acession Number AF279661) and T.gondii MECS forecast arrays(55. m04628,http://www.toxodb.org) as references,respectively.The EtMEC ORF sequence was predicted with this encoed amino acid by carrying out PSI-BLAST. EtMEC partical ORF sequence was amplified by RT- PCR with total RNA extracted from E.tenella the second generation merozoites as template.The authentic 3'-and 5'-terminal sequences of EtMEC gene were obtained by using the rapid amplification of cDNA end(RACE) techniques.The full length of EtMEC gene is 1625bp,which contains the full ORF in length of 1218bp and includes 5'-untranslated region(5'-UTR) and a 3'-untranslated region (3'-UTR),which are 27bp and 380bp,respectively.A DNA fragment in length of 1625bp contained the full ORF was amplified by RT-PCR using a pair of special primers which was designed based on EtMEC cDNA sequence.The sequencing result indicated this sequence included a complete ORF with 1218 bp which encoded 405 amino acids.Amino acid sequences alignment of EtMEC against those of MEC from other model organisms by Clustal W showed 24-45%identity in amino acid composition.There is a leader peptide fragment in EtMEC involved in a signal peptide in length of 24 amino acids(initiated from N-terminal) and an apicoplast targeted transit peptide with 128 amino acids by Signal P3.0 prediction. This revealed the fragment of 759bp encoding the mature protein of EtMEC was composed of 253 amino acids with a predicted molecular weight of 37.6kD. Phylogenetic analysis of MECs indicated that EtMEC was in the same cluster with T.gondii MEC.Predicted conformation of mature EtMEC is similar to T.gondii MEC.(2) The encoded region of EtMEC without N-terminal signal peptide was inserted into pET-32a(+) vector to construct the recombinant expresion vector,which was induced into E.coli Rosseta(DE3).The fusion protein was expressed successfully by inducing with IPTG.The result of SDS-PAGE indicated that the WM of the expressed protein was about 57.6 KD.Most of the recombinant protein was existed as the inclusion body.Using different temperature for optimizing condition,37℃is better than 16℃.The fusion protein was purified successfully by 6×His Resin column purification system.To determine the subcellar localization of EtMEC in Sporozoites,the EtMEC was located in the apicoplast of E.tenella by the immunofluorescenced visualization of confocal microscope.