Molecular And Biochemical Characterization Of B-Ketoacyl-ACP Reductase Of Eimeria Tenella

Coccidiosis is an important avian disease which is seriously harms the poultry production. The strategies of controlling this economically important disease rely heavily on chemoprophylaxis.Due to the increasing emergency of drug-resistant strains,people have to explore new target for anti-coccidials.Typeâ…¡fatty acid biosynthesis pathway(FASâ…¡) represents a potential target for the discovery of new anti-coccidials.We predicted the ORF sequence encoding theβ-ketoacyl- ACP reductase of Eimeria tenella(EtKR),one of the key enzymes in FASâ…¡by the bioinformatic analysis against Eimeria tenella gene database (www.sanger.ac.uk/Projects/E-tenella/),and the gene was cloned using the specific primers designed,from this predicted gene,the kinetic characterations of recombinant EtKR(rEtKR) were performed.The experiments include the following contents:(1) In this study,EtKR ORF sequence had been successfully predicted by Bioinformatic assembly.Based on the predicted sequence,EtKR partical ORF sequence was amplified by RT-PCR with total RNA extracted from E.tenella second generation merozoites as template.The authentic 3'-and 5'-terminal sequences of EtKR gene were obtained by using the rapid amplification of cDNA end(RACE) techniques.The full length of EtKR gene is 1799bp in which contains the full ORF in length of 1044bp,with a 5'-untranslated region(5'-UTR) and a 3'-untranslated region(3'-UTR),which is 247bp and 508bp,respectively..A DNA fragment in length of 1184bp contained the full ORF was amplified by RT-PCR using a pair of special primers which was designed based on EtKR cDNA sequence.The sequence result indicated this sequence included a complete ORF with 1044 bp which encoded 347 amino acids.Amino acid sequences alignment of EtKR against those of KR from other model organisms by Clustal W showed an identity of 35-50%identity in amino acid composition.Analysis by Signal P3.0 revealed that EtKR possess a leader peptide fragment in which in involved a signal peptide in length of 22 amino acids(initiated from N-terminal) and an apicoplast targeted transit peptide with 78 amino acids.This revealed fragment of 744bp encoding the mature protein of EtKR is composed of 247 amino acids with a predicted molecular weight of 24.9kD.In addition,a (Ser-Tyr-Lys) active site was observed,indicated that EtKR belongs to the family of short-chain dehydrogenases/redutase(SDR).Phylogenetic analysis of KRs indicated that EtKR was in the same cluster with P.falciparum KR(P.fKR).Predicted conformation of mature EtKR displayed typical Rossman-fold,similar to P.falciparum KR.(2) By the means of DNA recombination,encoding region of EtKR without N-terminal signal peptide and transit peptide was cloned into pET-32a(+) and/or pMAL-c2x vector, and transformed to E.coli BL21.The fusion protein was expressed by inducing with IPTG The result of SDS-PAGE indicated that the expression protein was about 45KD and 67 KD, respectively.Most of the recombinant protein in the pET-32a(+) existed in the inclusion body,the recombinant protein in the pMAL-c2x vector was soluble,and purified by Amylose Resin column purification system.To determine the subcellar localization of EtKR in Sporozoites,Polyclonal antibodies against EtKR were produced using BALB/c mice.The EtKR was located in the apicoplast of E.teneIla by the immunofluorescenced visualization of conforcal microscope.(3) The enzymatic characterization of EtKR was studied.The recombinant form(rEtKR) expressed in bacteria was not able to catalyse the reaction with the substrate of AcAcCoA and NADPH.However,it can oxidize NADH with a Km of 72.9/aM.We presumed that the reason of function inefficient of rEtKR possibly due to the inappropriate folding of rEtKR expressed in bacteria.Function of recombinant EtKR could be inhibited by hexachlorophene and the IC₅₀ for hexachlorophene was 20.9μM,suggesting that this enzyme may be explored as a potential and novel drug target.